Methods for Treating Central Nervous System Disorders Using VDAC Inhibitors

ABSTRACT

The present invention relates to use of small organic compounds interacting with the Voltage-Dependent Anion Channel (VDAC) for the treatment of diseases associated with central nervous system (CNS) disorders, including psychotic disorders, mood disorders, neurodegenerative diseases. In particular the present invention relates to the use of substituted piperazine- and piperidine-derivatives and pharmaceutical compositions comprising same for the treatment of psychotic disorders including schizophrenia, mood disorders, and neurodegenerative diseases.

This application claims the benefit of each of International PatentApplication No. PCT/IL2016/051020, filed Sep. 13, 2016, and U.S.Provisional Patent Application No. 62/397,991, filed Sep. 22, 2016, withthe entire content of each application being expressly incorporatedherein by reference thereto.

FIELD OF THE INVENTION

The present invention relates to use of small organic compoundsinteracting with the Voltage-Dependent Anion Channel (VDAC) for thetreatment of central nervous system (CNS) disorders and diseases. Inparticular the present invention relates to the use of substitutedpiperazine- and piperidine-derivatives and pharmaceutical compositionscomprising them for the treatment of psychotic disorders includingschizophrenia, mood disorders including bipolar disorder, andneurodegenerative diseases including Alzheimer's disease, Parkinson'sdisease and Amyotrophic Lateral Sclerosis (ALS).

BACKGROUND OF THE INVENTION

VDAC forms the main interface between mitochondrial and cellularmetabolisms by mediating the fluxes of ions, nucleotides and othermetabolites across the outer mitochondrial membrane (OMM)(Shoshan-Barmatz, V et al. 2010. Molecular Aspects of Medicine 31(3),227-286; Shoshan-Barmatz V and Ben-Hail D. 2012. Mitochondrion,12(1):24-34). VDAC has also been recognized as a key protein inmitochondria-mediated apoptosis. VDAC mediates the release ofapoptosis-inducing proteins from mitochondria to the cytosol andregulates apoptosis via interaction with pro- and anti-apoptoticproteins (Shoshan-Barmatz V et al. 2010, ibid; Shoshan-Barmatz V andGolan M. 2012. Current Medicinal Chemistry 19(5), 714-735).

Mitochondrial-bound hexokinase (HK), subtypes I and II (HK-I and HK-II),functions in the coupling of cytosolic glycolysis to mitochondrialoxidative phosphorylation. This is mediated via HK interaction withVDAC1. VDAC1-bound HK also prevents the release of pro-apoptoticfactors, and subsequent apoptosis accompanied with detachment of HK. HKdetachment from mitochondria has been observed in several pathologicalconditions, such as Alzheimer's Disease (Saraiva, L M et al. 2010. PLoSONE 5, e15230), Parkinson's disease (Okatsu, K et al. 2012. BiochemBiophys Res Commun 428, 197-202) and mood and psychotic disorders,including schizophrenia (Rezin G T et al., 2009. Neurochem Res 34,1021-1029; Regenold, W T ET AL., 2012. J. Psychiatr Res. 46, 95-104;Shan, D et al. 2014. Schizophr Res 154, 1-13).

Piperazine and piperidine are used as essential sub-structure motifs invarious drugs. Piperazine pyrrolidine-2,5-dione derivatives have alsobeen demonstrated as malic enzyme inhibitors (Zhang Y J et al. 2006.Bioorganic & Medicinal Chemistry Letters 16, 525-528).

A publication of the inventors of the present invention and co-workers,published after the priority date of the present invention, describescompounds that directly interact with VDAC1 and prevent VDAC1oligomerization, concomitant with an inhibition of apoptosis as inducedby various means and in various cell lines. The compounds protectedagainst apoptosis-associated mitochondrial dysfunction, restoringdissipated mitochondrial membrane potential, and thus cell energy andmetabolism, decreasing reactive oxidative species production, andpreventing detachment of hexokinase bound to mitochondria and disruptionof intracellular Ca²⁺ levels (Ben Hail D et al., 2016. J Biol Chem291(48), 24986-25003).

Central nervous system (CNS) disorders are diseases that can affect thebrain or spinal cord. The CNS disorders may be caused by trauma,infections, degeneration, structural defects, tumors, blood flowdisruption, autoimmunity, or strokes. There exists a wide range oftreatments for these disorders, such as surgery, rehabilitation, andmedications. Examples of CNS medications include analgesics,anticonvulsants, antipsychotics, sedatives, and tranquilizers. Despitetheir beneficial effects, CNS medications have the potential fordeveloping tolerance, dependence, or addiction. Psychiatric disorders,including mood disorders and schizophrenia, as well as neurodegenerativedisease, are devastating CNS associated diseases with no effective,side-effect free treatment.

Thus, there remains a need for improved treatments of CNS disorders,particularly psychiatric disorders and neurodegenerative diseases thatprovide increased efficacy and reduces or eliminates any potential sideeffects.

SUMMARY OF THE INVENTION

The present invention provides methods for treating diseases associatedwith CNS disorders, employing small organic compounds interacting withand inhibiting VDAC activities associated with apoptosis and energymetabolism, particularly inhibiting VDAC oligomerization and thedetachment of HK-I from mitochondria.

The present invention provides a method for treating CNS-associateddisease selected from the group consisting of neurodegenerative disease,mood disorder and psychotic disorder, comprising administering to asubject in need thereof a therapeutically effective amount of at leastone substituted piperazine and piperidine derivative of general formula(I) as defined hereinafter, including the stereoisomers, enantiomers,mixtures thereof and salts thereof.

The present invention is based in part on the unexpected discovery thatcompounds of general Formulae (I) inhibit the oligomerization ofmitochondrial Voltage-Dependent Anion Channel (VDAC) protein, and,furthermore, inhibit the detachment of hexokinase from the mitochondrialVDAC1, affecting energy metabolism and inhibiting apoptosis. Thecompounds are therefore suitable for the treatment of CNS-associateddisorders, particularly mood and psychotic disorders, includingschizophrenia. In addition the compounds are effective for the treatmentof neurodegenerative disease, particularly Alzheimer's disease,Parkinson disease and amyotrophic lateral sclerosis (ALS).

According to one aspect, the present invention provides a method fortreating CNS-associated disorder, the method comprising administering toa subject in need thereof a pharmaceutical composition comprising atherapeutically effective amount of at least one compound of the generalFormula (I):

wherein:

A is carbon (C) or nitrogen (N);

R³ is absent, or is selected from a hydrogen, an unsubstituted orsubstituted amide or a heteroalkyl group comprising 3-12 atoms apartfrom hydrogen atoms, wherein at least one of said 3-12 atoms is aheteroatom, selected from nitrogen, sulfur and oxygen; wherein when A isnitrogen (N), R³ is absent;

L¹ is absent or is an amino linking group —NR⁴—, wherein R⁴ is hydrogen,a C₁₋₅-alkyl, a C₁₋₅-alkylene or a substituted alkyl —CH₂R, wherein R isa functional group selected from the group consisting of hydrogen, halo,haloalkyl, cyano, nitro, hydroxyl, alkyl, alkenyl, aryl, alkoxyl,aryloxyl, aralkoxyl, alkylcarbamido, arylcarbamido, amino, alkylamino,arylamino, dialkylamino, diarylamino, arylalkylamino, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl and heteroaryl;

R¹ is an aromatic moiety, which is optionally substituted with one ormore of Z;

Z is independently at each occurrence a functional group selected fromthe group consisting of, hydrogen, halo, haloalkyl, haloalkoxy,perhaloalkoxy or C₁₋₂-perfluoroalkoxy, cyano, nitro, hydroxyl, alkyl,alkenyl, aryl, alkoxyl, aryloxyl, aralkoxyl, alkylcarbamido,arylcarbamido, amino, alkylamino, arylamino, dialkylamino, diarylamino,arylalkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,alkylcarbonyloxy, arylcarbonyloxy, carboxyl, alkoxycarbonyl,aryloxycarbonyl, sulfo, alkylsulfonylamido, alkylsulfonyl, arylsulfonyl,alkylsulfinyl, arylsulfinyl and heteroaryl;

L² is a linking group, such that when A is nitrogen (N), L² is a groupconsisting of 4-10 atoms, apart from hydrogen atoms, optionally forminga ring, whereof at least one of the atoms is nitrogen, said nitrogenforming part of an amide group; and when A is carbon (C), then L² isselected from C₁₋₄ alkylene or a group consisting of 4-10 atoms, apartfrom hydrogen atoms, optionally forming a ring, whereof at least one ofthe atoms is nitrogen, said nitrogen forming part of an amide group; and

R² is a phenyl or a naphthyl, optionally substituted with halogen;

The invention also relates to the stereoisomers, enantiomers, mixturesthereof and salts thereof, of the compounds of general Formula I, andall their derivatives, according to the invention.

According to some embodiments, the psychotic disorder is selected fromthe group consisting of, but not limited to, schizophrenia, autismspectrum disorder and anorexia nervosa. Each possibility represents aseparate embodiment of the present invention. According to certainexemplary embodiments, the psychotic disorder is schizophrenia.

According to certain embodiments, the mood disorder is selected from thegroup consisting of, but not limited to, bipolar disorder, majordepressive disorder, persistent depressive disorder (also known asdysthymia) and anxiety disorder. Each possibility represents a separateembodiment of the present invention.

According to certain exemplary embodiments, the mood disorder is bipolardisorder.

According to certain embodiments, the neurodegenerative disease isselected from the group consisting of, but not limited to, Alzheimer'sdisease, Parkinson's disease and Amyotrophic Lateral Sclerosis (ALS).Each possibility represents a separate embodiment of the presentinvention.

According to certain exemplary embodiments, the neurodegenerativedisease is Alzheimer's disease.

According to certain additional or alternative embodiments, theneurodegenerative disease is Parkinson's disease.

According to certain additional or alternative embodiments, theneurodegenerative disease is ALS.

According to certain exemplary embodiments, the compounds of the presentinvention are administered to the subject in need thereof within apharmaceutical composition further comprising a pharmaceuticallyacceptable excipient, diluents or carriers.

According to some embodiments, the pharmaceutical composition furthercomprises at least one additional active agent.

Other aspects and embodiments of the present invention will becomeapparent to the skilled person from the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates a representative chromatogram and respective massspectra of two peaks of interest relating to Intermediate 1.

FIG. 2a demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to the compound of Formula 2.

FIG. 2b demonstrates a representative NMR spectrum relating to thecompound of Formula 2.

FIG. 3 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 2.

FIG. 4a demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to the compound of Formula 1.

FIG. 4b demonstrates a representative NMR spectrum relating to thecompound of Formula 1.

FIG. 5 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 3.

FIG. 6 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 4.

FIG. 7 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 5.

FIG. 8 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 6.

FIG. 9 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 7.

FIG. 10 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 8.

FIG. 11a demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to the compound of Formula 3.

FIG. 11b demonstrates a representative NMR spectrum in deuterated DMSOrelating to the compound of Formula 3.

FIG. 11c demonstrates a representative NMR spectrum in deuterated DMSOand deuterated water (D₂O) relating to the compound of Formula 3.

FIGS. 12a and 12b demonstrate representative NMR spectra in deuteratedDMSO relating to separated single enantiomers of the compound of Formula1 (identified as BGD-4-1 and VBIT-4-2, respectively).

FIG. 13 shows immunoblots and quantitative data of selenite-induced HK-Irelease to the cytosol and inhibition by VBIT-4, presented in relativeunits (RU).

FIG. 14a demonstrates the effect of compound of Formula 1 on learningand memory of transgenic mice with Alzheimer's disease like symptomsusing Radial Arm Water Maze test; number of errors is demonstrated asfunction of learning blocks.

FIG. 14b demonstrates the effect of compound of Formula 1 on learningand memory of transgenic mice with Alzheimer's disease like symptomsusing Radial Arm Water Maze test; total time per test is demonstrated asfunction of learning blocks.

FIG. 15a shows cross section of wild type (WT) and transgenic mice withAD-like disease (TG).

FIG. 15b shows immuno-staining of brain sections from the WT and TG micepresented in FIG. 15A with anti-Aβ or anti-VDAC1 antibodies.

FIGS. 16a and 16b shows memory function of wild type mice (WT) andtransgenic mice with AD-like disease (TG) untreated (TG V) or treatedwith VBIT-4 (TG T).

DETAILED DESCRIPTION

The present invention relates to the use of piperazine and/or piperidinederivatives interacting with VDAC, acting as inhibitors of, inter alia,VDAC oligomerization and the detachment of mitochondrial VDAC1-bound HK.

A critical feature of brain energy metabolism is attachment to the outermitochondrial membrane (OMM), via binding to VDAC, of hexokinase-I andhexokinase (HK-I and HK-II, respectively), initial and rate-limitingenzymes of glycolysis. HK-I and HK-II attachment to the OMM greatlyenhances their enzymatic activity and couples cytosolic glycolysis tomitochondrial oxidative phosphorylation, through which the cell producesmost of its adenosine triphosphate (ATP). Mitochondrial attachment ofHK-I or HK-II is also important to the survival of neurons and othercells through prevention of apoptosis and oxidative damage.

The present invention discloses for the first time the use ofpiperazine- and piperidine-derivatives of Formula (I) for treatingcentral nervous system (CNS) disease, particularly psychotic disorders,mood disorders and neurodegenerative diseases. Without wishing to bebound by certain specific theory or mechanism of action, inhibition ofmitochondrial HK-1 and/or HK-II by the compounds of the invention leadsto normal energy metabolism within brain and neuronal cells, whichcontributes to prevention or reduction of symptoms of CNS-associateddiseases.

Mitochondrial bound HK-I and HK-II also protects against apoptosis andrelease of reactive oxygen species (ROS) from the mitochondria to thecytosol.

HK-I and HK-II bound to the mitochondria are less sensitive toinhibition by their product glucose-6-phosphate.

Definitions

The term “VDAC” as used herein, unless the context explicitly dictatesotherwise, refers to Voltage-Dependent Anion Channel proteins of ahighly conserved family of mitochondrial porins. The term refers to allVDAC isoforms, e.g. to isoform VDAC1, to isoform VDAC2, or to isoformVDAC3.

The terms “hexokinase” or “HK”, as used herein, unless the contextexplicitly dictates otherwise, refer to hexokinase enzyme, to all itsisoforms, e.g. to isoform HK-I, and HK-II, HK-III and HK-IV. Hexokinaseisoforms may also refer to herein as HK-1 HK-2, HK-3 and HK-4.

The terms “central nervous system (CNS) disease”, “CNS disorder”“CNS-associated disease or “CNS associated disorder” are used hereininterchangeably and refer to one of a group of neurological disordersthat affect the structure or function of the brain or spinal cord, whichcollectively form the central nervous system. According to certainembodiments, the CNS disease is selected from the group consisting ofpsychotic disorders, mood disorder and neurodegenerative disease.According to some embodiments, the psychotic disorder is schizophrenia.According to some embodiments, the mood disorder is selected from thegroup consisting of bipolar disorder, depressive disorder and anxietydisorder. According to some embodiments, the neurodegenerative diseaseis selected from the group consisting of Alzheimer disease and Parkinsondisease.

As used herein, the term “schizophrenia” refers to a chronicdebilitating disorder, characterized by a spectrum of psychopathology,including positive symptoms such as aberrant or distorted mentalrepresentations (e.g., hallucinations, delusions), negative symptomscharacterized by diminution of motivation and adaptive goal-directedaction (e.g., anhedonia, affective flattening, avolition), and cognitiveimpairment.

“Mood disorder” as used herein refers to a group of diagnoses in theDiagnostic and Statistical Manual of Mental Disorders (DSM)classification system where a disturbance in the person's mood ishypothesized to be the main underlying feature. The classification isknown as mood (affective) disorders in International Classification ofDiseases (ICD). Mood disorders fall into the basic groups of elevatedmood, such as mania or hypomania; depressed mood, of which thebest-known and most researched is major depressive disorder (MDD)(commonly called clinical depression, unipolar depression, or majordepression); and moods which cycle between mania and depression, knownas bipolar disorder (BD). There are several sub-types of depressivedisorders or psychiatric syndromes featuring less severe symptoms suchas dysthymic disorder (similar to but milder than MDD) and cyclothymicdisorder (similar to but milder than BD). Mood disorders may also besubstance-induced or occur in response to a medical condition.

The terms “bipolar disorder”, “manic-depressive disorder” “bipolarism”or “manic depression” refer to a psychiatric diagnosis that describes acategory of mood disorders defined by the presence of one or moreepisodes of abnormally elevated mood clinically referred to as mania or,if milder, hypomania. Individuals who experience manic episodes alsocommonly experience depressive episodes or symptoms, or mixed episodesin which features of both mania and depression are present at the sametime. These episodes are usually separated by periods of “normal” mood,but in some individuals, depression and mania may rapidly alternate,known as rapid cycling. Extreme manic episodes can sometimes lead topsychotic symptoms such as delusions and hallucinations. The disorderhas been subdivided into bipolar I, bipolar II, cyclothymia, and othertypes, based on the nature and severity of mood episodes experienced;the range is often described as the bipolar spectrum. The term “mania”or “manic periods” or other variants refers to periods where anindividual exhibits some or all of the following characteristics: racingthoughts, rapid speech, elevated levels of activity and agitation aswell as an inflated sense of self-esteem, euphoria, poor judgment,insomnia, impaired concentration and aggression.

The term “depression” as used herein includes, but is not limited to,major depressive episodes in the context of major depressive disorder orbipolar disorder, schizoaffective disorder and other psychiatric statesthat are characterized by depressed mood and/or feelings of sadness,despair, discouragement, “blues”, melancholy, feelings of lowself-esteem, guilt and self-reproach, withdrawal from interpersonalcontact, and somatic symptoms such as eating and sleep disturbances.Depression may be also associated with other psychiatric disorders, withgeneral medical conditions or with alcohol or drug abuse. Examples ofother conditions that may be associated with depression include, but arenot limited to, dysthymia, posttraumatic stress disorder, schizophrenia,post-partum depression, eating disorders including anorexia nervosa andbulimia, anxiety disorders, Parkinson's disease, Alzheimer's disease,fibromyalgia and chronic fatigue syndrome.

Anxiety disorders include, but are not limited to, obsessive compulsivedisorder, generalized anxiety disorder, panic disorder and socialphobia.

As used herein, the terms “Alzheimer disease”, “Alzheimer's disease” and“AD” are used herein interchangeably, and refer to all types and stagesof the disease including preclinical and prodromal stages; also known as“dementia of the Alzheimer type. Alzheimer's disease is characterized bymemory deficits in its early phase. Later symptoms include impairedjudgment, disorientation, confusion, behavior changes, trouble speaking,and motor deficits. Histologically. AD is characterized by beta-amyloidplaques and tangles of protein tau.

The terms “Parkinson's disease” or PD refer to a disease that belongs toa group of chronic and progressive conditions called movement disorders.PD is characterized by muscle rigidity, tremor, a slowing of physicalmovement (bradykinesia) and, in extreme cases, a loss of physicalmovement (akinesia). The primary symptoms are the results of decreasedstimulation of the motor cortex by the basal ganglia, normally caused bythe insufficient formation and action of dopamine, which is produced inthe dopaminergic neurons of the brain. Secondary symptoms may includehigh level cognitive dysfunction and subtle language problems.

The terms “Amyotrophic lateral sclerosis” and “ALS” refer to aprogressive, fatal, neurodegenerative disease characterized by adegeneration of motor neurons, the nerve cells in the central nervoussystem that control voluntary muscle movement. ALS is also characterizedby neuronal degeneration in the entorhinal cortex and hippocampus,memory deficits, and neuronal hyperexcitability in different brain areassuch as the cortex.

As used herein, the terms “inhibition” or “inhibiting” with regard toinhibiting the detachments of mitochondrial VDAC-bound HK refer toinhibiting the detachment of HK from the mitochondrial VDAC such that HKamount in a cell cytosol is reduced upon administering of a compound ofthe invention compared to its amount in a corresponding cell withoutaddition of the compound. According to certain embodiments, thecytosolic HK amount is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99% or 100% compared to its amount in acorresponding cell with the addition of an inhibitory compound. Eachpossibility represents a separate embodiment of the present invention.

The term “therapeutically effective amount” as used herein with regardto a compound of the invention is an amount of a compound that, whenadministered to a subject will have the intended therapeutic effect,e.g. improving symptom(s) associated with a CNS disorder. The fulltherapeutic effect does not necessarily occur by administering of onedose, and may occur only after administering of a series of doses. Thus,a therapeutically effective amount may be administered in one or moredoses. The precise effective amount needed for a subject will dependupon, for example, the subject's weight, health and age, the nature ofthe CNS disorder and extent of the symptoms of the specific CNS disorder(such as schizophrenia and additional psychotic disorders, bipolardisorder and additional mood disorders, Alzheimer's Disease (AD) andParkinson's), and optionally, the combination of the compounds of theinvention with additional therapeutics, and the mode of administered.

The term “preventing” as used herein means causing the clinical symptomsof the disease state not to develop in a subject that may be exposed toor predisposed to the disease state, but does not yet experience ordisplay symptoms of the disease state.

The term “treating” as used herein refers to inhibiting the diseasestate, i.e., arresting the development of the disease state or itsclinical symptoms, or relieving the disease state, i.e., causingtemporary or permanent regression of the disease state or its clinicalsymptoms. The term is interchangeable with any one or more of thefollowing: abrogating, ameliorating, inhibiting, attenuating, blocking,suppressing, reducing, halting, alleviating or preventing symptomsassociated with the disease.

According to one aspect, the present invention provides a method fortreating CNS-associated disorder, the method comprising administering toa subject in need thereof at least one substituted piperazine- andpiperidine-derivative of general Formula (I):

wherein:

A is carbon (C) or nitrogen (N);

R³ is absent, a hydrogen, an unsubstituted or substituted amide or aheteroalkyl group comprising 3-12 atoms apart from hydrogen atoms,wherein at least one of said 3-12 atoms is a heteroatom, selected fromnitrogen, sulfur and oxygen; wherein when A is nitrogen (N), R³ isabsent;

L¹ is absent or is an amino linking group —NR⁴—, wherein R⁴ is hydrogen,a C₁₋₅-alkyl, a C₁₋₅-alkylene or a substituted alkyl —CH₂R, wherein R isa functional group selected from the group consisting of hydrogen, halo,haloalkyl, cyano, nitro, hydroxyl, alkyl, alkenyl, aryl, alkoxyl,aryloxyl, aralkoxyl, alkylcarbamido, arylcarbamido, amino, alkylamino,arylamino, dialkylamino, diarylamino, arylalkylamino, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl and heteroaryl; preferably R⁴ is hydrogen;

R¹ is an aromatic moiety, preferably phenyl, which may be substitutedwith one or more of Z;

Z is independently at each occurrence a functional group selected fromthe group consisting of, hydrogen, halo, haloalkyl, haloalkoxy,perhaloalkoxy or C₁₋₂-perfluoroalkoxy, cyano, nitro, hydroxyl, alkyl,alkenyl, aryl, alkoxyl, aryloxyl, aralkoxyl, alkylcarbamido,arylcarbamido, amino, alkylamino, arylamino, dialkylamino, diarylamino,arylalkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,alkylcarbonyloxy, arylcarbonyloxy, carboxyl, alkoxycarbonyl,aryloxycarbonyl, sulfo, alkylsulfonylamido, alkylsulfonyl, arylsulfonyl,alkylsulfinyl, arylsulfinyl and heteroaryl; preferably Z isC₁₋₂-perfluoroalkoxy; preferably R¹ is a phenyl and Z istrifluoromethoxy; preferably R¹ is a phenyl substituted with onetrifluoromethoxy, most preferably at the para position;

L² is a linking group, such that when A is nitrogen (N), L² is a groupconsisting of 4-10 atoms (apart from hydrogen atoms), optionally forminga ring, whereof at least one of the atoms is nitrogen, said nitrogenforming part of an amide group; preferably said linking group isselected from the group consisting of an C₄₋₆-alkylamidylene and apyrrolidinylene, said linking group optionally substituted with one ortwo of alkyl, hydroxy, oxo or thioxo group; most preferably L² isselected from butanamidylene, N-methylbutanamidylene,N,N-dimethylbutanamidylene, 4-hydroxybutanamidylene(HO—CH₂—C*H—CH₂—C(O)NH—, wherein the asterisk denotes attachment point),4-oxobutanamidylene, 4-hydroxy-N-methylbutanamidylene,4-oxo-N-methylbutanamidylene, 2-pyrrolidonyl, pyrrolidine-2,5-dionylene,5-thioxo-2-pyrrolidinonylene and 5-methoxy-2-pyrrolidinonylene;

and when A is carbon (C), then L² is either as defined for L² when A isnitrogen (N) or C₁₋₄ alkylene; L² is preferably methylene (—CH₂—);

R² is a phenyl or a naphthyl, optionally substituted with halogen,preferably when R² is a phenyl it is substituted with halogen,preferably chlorine, at the para position, preferably when R² isnaphthyl, L² is an alkylene group, preferably —CH₂—;

with a proviso that when A is carbon (C), L¹ is —NR⁴—, R⁴ is hydrogen,and R² is phenyl substituted with chlorine, then L² is notpyrrolidine-2,5-dione.

In some embodiments, R³ is hydrogen or heteroalkyl group comprising 3-12atoms apart from hydrogen atoms, wherein at least one of said 3-12 atomsis a heteroatom, selected from nitrogen, sulfur and oxygen. In otherembodiments (i.e., when A is nitrogen), R³ is absent.

In some embodiments, R⁴ is hydrogen.

In some embodiments, R¹ is a phenyl substituted trifluoromethoxy. Insome embodiments, R¹ is a phenyl substituted with one trifluoromethoxy.In some embodiments, R¹ is a phenyl substituted with onetrifluoromethoxy at the para position. In some embodiments, R¹ is phenyl

In some embodiments, L² is a linking group, consisting of 4-10 atoms(apart from hydrogen atoms), optionally forming a ring, whereof at leastone of the atoms is nitrogen, said nitrogen forming part of an amidegroup; preferably said linking group is selected from the groupconsisting of an C₄₋₆-alkylamidylene and a pyrrolidinylene, said linkinggroup optionally substituted with one or two of alkyl, hydroxy, oxo orthioxo group; most preferably L² is selected from butanamidylene,N-methylbutanamidylene, N,N-dimethylbutanamidylene,4-hydroxybutanamidylene (HO—CH₂—C*H—CH₂—C(O)NH-wherein the asteriskdenotes attachment point), 4-oxobutanamidylene,4-hydroxy-N-methylbutanamidylene, 4-oxo-N-methylbutanamidylene,2-pyrrolidonyl, pyrrolidine-2,5-dionylene, 5-thioxo-2-pyrrolidinonyleneand 5-methoxy-2-pyrrolidinonylene; or L² is C₁₋₄ alkylene, preferablymethylene (—CH₂—);

The term “pyrrolidinylene” refers to a pyrrolidine ring as a bivalentsubstituent. Pyrrolidinylene include unsubstituted and substitutedrings, such as, but not limited to, pyrrolidine-2-5-dione,2-pyrrolidinone, 5-thioxo-2-pyrrolidinone, 5-methoxy-2-pyrrolidinone andthe like.

In one embodiment, when A is nitrogen (N), the linking group L² isselected from the group consisting of an C₄₋₆-alkylamidylene and apyrrolidinylene, said linking group optionally substituted with one ortwo of alkyl, hydroxy, oxo or thioxo group. For example, L² may bebutanamidylene, N-methylbutanamidylene, N,N-dimethylbutanamidylene,4-hydroxybutanamidylene, 4-oxobutanamidylene,4-hydroxy-N-methylbutanamidylene, 4-oxo-N-methylbutanamidylene,2-pyrrolidonyle, pyrrolidine-2,5-dionylene, 5-thioxo-2-pyrrolidinonyleneor 5-methoxy-2-pyrrolidinonylene. Preferably, when L² is butanamidylene,N-methylbutanamidylene, N,N-dimethylbutanamidylene,4-hydroxybutanamidylene, 4-oxobutanamidylene,4-hydroxy-N-methylbutanamidylene or 4-oxo-N-methylbutanamidylene, thenpreferably the carbon in third position (C) of the butanamide moiety isbonded to the nitrogen (N) of the piperazine ring or the piperidine ringand the nitrogen (N) of the butanamide moiety is bonded to R². Forexample, when L² is 2-pyrrolidone, pyrrolidine-2,5-dione,5-thioxo-2-pyrrolidone or 5-methoxy-2-pyrrolidone, then preferably acarbon (C) of the pyrrolidine moiety is bonded to the nitrogen (N) ofthe piperazine ring or the piperidine ring and the nitrogen (N) of thepyrrolidine moiety is bonded to R².

In another embodiment, A is carbon (C), R³ is heteroalkyl and L² ismethylene.

The invention also relates to the stereoisomers, enantiomers, mixturesthereof, and salts, particularly the physiologically acceptable salts,of the compounds of general Formula (I) according to the invention.

According to certain embodiments, the at least one substitutedpiperazine- and piperidine-derivative is of general Formula Ia:

wherein:

A, R³, Z and L¹ are as previously defined in reference to compound ofFormula (I); preferably A is nitrogen (N);

L²′ is a linking group selected from the group consisting of anC₄-alkylamidylene, C₅-alkylamidylene and C₆-alkylamidylene, optionallysubstituted with one or two of alkyl, hydroxy, oxo or thioxo group;preferably L²′ is selected from butanamidylene, N-methylbutanamidylene,N,N-dimethylbutanamidylene, 4-hydroxybutanamidylene,4-oxobutanamidylene, 4-hydroxy-N-methylbutanamidylene or4-oxo-N-methylbutanamidylene; most preferably L²′ is4-hydroxybutanamidylene; wherein preferably the carbon (C) at position 3of the alkyl moiety of alkylamidylene L²′ is bonded to the nitrogen (N)of the piperazine ring or of the piperidine ring, and the nitrogen (N)of the butanamide moiety is bonded to the phenyl group; preferablyL^(2′) is HO—CH₂—C*H—CH₂—C(O)NH—, wherein the asterisk denotesattachment point;

Y is halogen, preferably chlorine, e.g. at the para position;

or an enantiomer, diastereomer, mixture or salt thereof.

According to certain embodiments, the substituted piperazine- andpiperidine-derivative is of general Formula (Ib):

wherein:

A, R³, and Z are as previously defined in reference to the compound ofFormula (I); preferably A is nitrogen (N);

L¹ is absent;

L^(2″) is a pyrrolidinylene linking group, optionally substituted withone or two of alkyl, hydroxy, oxo or thioxo group, preferably L^(2″) isselected from 2-pyrrolidonylene, pyrrolidine-2,5-dionylene,5-thioxo-2-pyrrolidinonylene and 5-methoxy-2-pyrrolidinonylene; mostpreferably L^(2″) is pyrrolidine-2,5-dionylene; wherein preferably acarbon (C) at position 4 or the carbon (C) at position 3 of thepyrrolidinyl moiety L^(2″) is bonded to the nitrogen (N) of thepiperazine ring or the piperidine ring and the nitrogen (N) of thepyrrolidinyl moiety is bonded to the phenyl group substituted with Y;and

Y is halogen, preferably chlorine, e.g. at the para position.

According to certain embodiments, the substituted piperazine- andpiperidine-derivative is of general Formula (Ic):

wherein:

A, R³, and Z are as previously defined in reference to the compounds ofgeneral Formula (I);

L¹ is —NH—; and

Y¹ and Y² are each independently absent or a halogen;

or an enantiomer, diastereomer, mixture or salt thereof.

Preferred compounds of Formula (Ic) are those wherein R³ is—C(O)NHCH₂C(O)OH group, and/or wherein Z is C₁₋₂-alkoxy or halogenatedC₁₋₂-alkoxy, e.g. C₁₋₂-perfluoroalkoxy.

According to certain embodiments, the substituted piperazine- andpiperidine-derivatives is of general Formula (Id):

wherein

L² is selected from the group consisting of an C₄₋₆-alkylamidylene (e.g.HO—CH₂—C*H—CH₂—C(O)NH—, wherein the asterisk denotes attachment point),and a pyrrolidinylene (e.g. pyrrolidin-2,5-dionylene), optionallysubstituted with one or two of alkyl, hydroxy, oxo or thioxo group; and

Z is haloalkoxy, e.g. C₁₋₂-perfluoroalkoxy, and Y is halogen.

The invention also relates to the stereoisomers, enantiomers, mixturesthereof and salts thereof, of the compounds of general Formulae (Ia),(Ib), (Ic), and (Id), according to the invention.

Table 1 provides non-limiting examples of compound of general Formula(I). It includes compounds as follows:

-   N-(4-chlorophenyl)-4-hydroxy-3-(4-(4-(trifluoromethoxy)phenyl)-piperazin-1-yl)butanamide    (Formula 1);-   1-(4-chlorophenyl)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)pyrrolidine-2,5-dione    (Formula 2);-   1-(naphthalen-1-yl)methyl)-4-(phenylamino)-piperidine-4-carbonyl)glycine    (Formula 3);-   1-(4-chlorophenyl)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)pyrrolidin-2-one    (Formula 4);-   1-(4-chlorophenyl)-5-thioxo-3-(4-(4-(trifluoro-methoxy)phenyl)piperazin-1-yl)pyrrolidin-2-one    (Formula 5);-   1-(4-chlorophenyl)-5-methoxy-4-(4-(4-(trifluoromethoxy)phenyl)-piperazin-1-yl)pyrrolidin-2-one    (Formula 6);-   1-(4-chlorophenyl)-5-thioxo-4-(4-((4-(trifluoromethoxy)phenyl)amino)piperidin-1-yl)pyrrolidin-2-one    (Formula 7);-   4-(4-chlorophenyl)-4-oxo-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanamide    (Formula 8); and-   N-(4-chlorophenyl)-4-hydroxy-N-methyl-3-(4-(4-(trifluoro-methoxy)phenyl)piperazin-1-yl)butanamide    (Formula 9).

TABLE 1 examples of compound of general Formula (I) Formula Description# Structure (according to general Formula (I)) 1

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is phenyl substitutedwith one trifluoromethoxy, L² is 4- hydroxybutanamidylene, the 3^(rd)carbon (C) of the butanamide moiety is bonded to the nitrogen (N) of thepiperazine ring, the nitrogen (N) of the butanamide moiety is bonded toR² and R² is a phenyl substituted with chlorine at the para position[also identified herein as VBIT-4 or as BGD-4] 2

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is phenyl substitutedwith one trifluoromethoxy, L² is pyrrolidine-2,5-dione, the carbon (C)at position 3 of the pyrrolidine moiety is bonded to the nitrogen (N) ofthe piperazine ring, the nitrogen (N) of the pyrrolidine moiety isbonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-3 or as BGD-3] 3

A is carbon (C), R³ is —C(O)NHCH₂C(O)OH group; L¹ is —NH—, R¹ is aphenyl, L² is methylene and R² is a naphthyl [also identified herein asVBIT-12] 4

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is a phenylsubstituted with one trifluoromethoxy; L² is 2- pyrrolidine, the carbon(C) at position 3 of the pyrrolidine moiety is bonded to the nitrogen(N) of the piperazine ring, the nitrogen (N) of the pyrrolidone moietyis bonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-5] 5

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is a phenylsubstituted with one trifluoromethoxy, L² is 5- thioxo-2-pyrrolidone,the carbon (C) at position 3 of the pyrrolidine moiety is bonded to thenitrogen (N) of the piperazine ring, the nitrogen (N) of the pyrrolidinemoiety is bonded to R² and R² is a phenyl substituted with chlorine atthe para position [also identified herein as VBIT-6] 6

A is carbon (C), R³ is hydrogen, L¹ is —NH—, R¹ is a phenyl substitutedwith one trifluoromethoxy, L² is 5- methoxy-2-pyrrolidinone, the carbon(C) at position 4 of the pyrrolidine moiety is bonded to the nitrogen(N) of the piperidine ring, the nitrogen (N) of the pyrrolidine moietyis bonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-9] 7

A is carbon (C), R³ is hydrogen, L¹ is —NH—, R¹ is a phenyl substitutedwith one trifluoromethoxy, L² is 5- thioxo-2-pyrrolidone, the carbon (C)at position 3 of the pyrrolidine moiety is bonded to the nitrogen (N) ofthe piperidine ring, the nitrogen (N) of the pyrrolidine moiety isbonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-10] 8

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is phenyl substitutedwith one trifluoromethoxy, L² is 4- oxobutanamide, the 3^(rd) carbon (C)of the butanamide moiety is bonded to the nitrogen (N) of the piperazinering, the 4^(th) carbon (C) of the butanamide moiety is bonded to R² andR² is a phenyl substituted with chlorine at the para position [alsoidentified herein as VBIT-7] 9

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is phenyl substitutedwith one trifluoromethoxy, L² is 4- hydroxy-N-methylbutanamide, the3^(rd) carbon (C) of the butanamide moiety is bonded to the nitrogen (N)of the piperazine ring, the nitrogen (N) of the butanamide moiety isbonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-8]

Some terms used herein to describe the compounds according to theinvention are defined more specifically below.

The term halogen denotes an atom selected from among F, Cl, Br and I,preferably Cl and Br.

The term heteroalkyl as used herein in reference to R³ moiety of thegeneral Formulae (I), (Ia), (Ib), (Ic), (Id), and (IIa), refers to asaturated or unsaturated group of 3-12 atoms (apart from hydrogenatoms), wherein one or more (preferably 1, 2 or 3) atoms are a nitrogen,oxygen, or sulfur atom, for example an alkyloxy group, as for examplemethoxy or ethoxy, or a methoxymethyl-, nitrile-,methylcarboxyalkylester- or 2,3-dioxyethyl-group; preferably heteroalkylgroup is a chain comprising an alkylene, and at least one of acarboxylic acid moiety, a carbonyl moiety, an amine moiety, a hydroxylmoiety, an ester moiety, an amide moiety. The term heteroalkyl refersfurthermore to a carboxylic acid or a group derived from a carboxylicacid as for example acyl, acyloxy, carboxyalkyl, carboxyalkylester, suchas for example methylcarboxyalkylester, carboxyalkylamide,alkoxycarbonyl or alkoxycarbonyloxy; preferably the term refers to—C(O)NHCH₂C(O)OH group.

The term C_(1-n)-alkyl, wherein n may have a value as defined herein,denotes a saturated, branched or unbranched hydrocarbon group with 1 ton carbon (C) atoms. Examples of such groups include methyl, ethyl,n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl,iso-pentyl, neo-pentyl, tert-pentyl, n-hexyl, iso-hexyl, etc.

The term C₁₋₄-alkyl denotes a saturated, branched or unbranchedhydrocarbon group with 1 to 4 carbon (C) atoms.

The term C_(1-n)-alkoxy, wherein n may have a value as defined herein,denotes an alkyl group as defined herein, bonded via —O— (oxygen)linker.

The term “C_(1-n) alkylene”, wherein n may have a value as definedherein, denotes an alkylene group of saturated hydrocarbons substituentswith the general formula C_(n)H_(2n). Generally, n is a positiveinteger. For example, C₁ alkylene refers to methylene (—CH₂—), C₃alkylene refers to C₃H₆, which may be n-propylene (—CH₂CH₂CH₂—) orisopropylene (—CH(CH₃)CH₂— or —CH₂CH(CH₃)—). Preferably the term refersto an unbranched n-alkylene.

The term C_(1-n)-perfluoroalkoxy, wherein n may have a value as definedherein, denotes an alkoxy group with hydrogen atoms substituted byfluorine atoms.

The term C_(1-m)-alkylamidyl, wherein m may have a value as definedherein, denotes a group comprising 1 to m carbon (C) atoms and an amidegroup formed by either C_(m-a)alkyl-COOH and H₂N-C_(a)alkyl, orC_(m-a)alkyl-NH₂ and HOOC-C_(a)alkyl, wherein a is smaller than or equalto m. Similarly, the terms C₄-alkylamidylene, C₅-alkylamidylene andC₆-alkylamidylene refer to divalent C_(m)-alkylamidyl groups, wherein mis either 4, 5, or 6, respectively.

Certain compounds of the general Formula (I), wherein L¹ is absent, andA is nitrogen, may be prepared by coupling an aryl halide of the formulaR¹—X, wherein X is a halogen, preferably bromide, with a mono-protectedpiperazine, e.g. with BOC-protected piperazine, and upon deprotection,reacting with a L²-linker precursor reactive with secondary amines, andsubsequent amidation or transamidation of the L²-linker precursor moietywith a suitable amine of the formula (Y)R²—NH₂. The L²-linker precursormay be an unsaturated C₄₋₆ carboxylic derivative compound, e.g. anunsaturated C₄₋₆ lactone, or a β-, γ-, δ- or ε-unsaturated linear esterof the C₄₋₆ carboxylic acid and a suitable alcohol, e.g. C₁₋₆ alcohol.Alternatively, the deprotected R¹-piperazine may be reacted with asuitable N—R²-pyrrolidenone or N—R²-pyrrolidinene-dione, prepared bygenerally known methodology (e.g. in Synthesis, anticonvulsant activityand 5-HT1A, 5-HT2A receptor affinity of newN-[(4-arylpiperazin-1-yl)-alkyl] derivatives of 2-azaspiro[4.4]nonaneand [4.5]decane-1,3-dione, by Obniska, J et al. 2006. European Journalof Medicinal Chemistry 41(7), 874-881).

Compounds of general Formulae (Ia) and (Ib) may be prepared according toa Method (a) shown in Schemes 1 to 3, starting from a compound ofgeneral Formula A, wherein Z is as hereinbefore defined.

Compounds of general Formula C are obtained by reacting a compound ofgeneral Formula A with a piperazine in which one of the nitrogens isprotected with a protecting group, e.g. tert-butyloxycarbonyl protectinggroup (BOC group). The starting compounds of general Formula A is eithercommercially obtainable or may be prepared by using known methods fromcommercially obtainable compounds. The carbon-nitrogen (C—N) couplingreaction is carried out in the presence of a palladium catalyst, such astris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃) that is particularlysuitable. The reaction is carried out in a presence of a bidentatephosphine ligand and a base. Suitable bidentate phosphine ligands arediphenyl-phosphinobinapthyl (BINAP) and diphenylphosphinoferro-cene(DPPF), while BINAP is particularly preferred. Suitable bases includesodium tert-butoxide, potassium tert-butoxide, lithiumbis(trimethylsilyl)-amide, while sodium tert-butoxide is particularlysuitable. The reaction is carried out in a suitable aprotic solvent suchas toluene, tetrahydrofuran (THF), dioxane, but preferably toluene,under nitrogen atmosphere, and at a temperature between 55° C. and 110°C., preferably at a temperature of 65° C. and 110° C., most preferably80° C. and 110° C. Upon completion of the reaction the solvent isevaporated to provide crude compound of Formula C as a residue that maybe used for the next step without further purification.

Compounds of general Formula D are obtained by removal of the protectinggroup, e.g. BOC, in the compound of general Formula C, which can beaccomplished with strong acids such as trifluoroacetic acid, neat or indichloromethane, or with concentrated HCl in methanol or indichloromethane (DCM), while concentrated HCl in DCM is preferred. Thereaction is preferably carried out at room temperature. Upon completionof the reaction the organic phase is discarded and the aqueous phaseevaporated to dryness. The residue is dissolved in a base and a suitablesolvent, such as DCM, dichloroethane or 2-methyltetrahydrofuran (MeTHF),NaOH (2.0 M) and DCM are preferred. Upon completion of the reaction, theorganic solvent phase, e.g. DCM, is collected and concentrated to yieldthe crude product of general Formula D that may be used for the nextstep without further purification.

Compounds of general Formula (Ia) and (Ib) are obtained from compoundsof general Formula D. For example, certain preferred compounds ofgeneral Formula (Ia) may be obtained according to Scheme 2, by reactinga compound of general Formula D with a suitable lactone, e.g.2-furanone, to yield a compound of general Formula E, which is reactedwith a suitable aminophenyl to yield a compound of general Formula (Ia).

For example, certain preferred compounds of general Formula (Ib) may beobtained according to Scheme 3, by reacting a compound of generalFormula D with a suitable pyrrole-dione, e.g1-Phenyl-1H-pyrrole-2,5-dione, which is commercially available or mayreadily be prepared by methods familiar to those skilled in the art, toyield a compound of general Formula (Ib).

Certain compounds of the general Formula (I) wherein R³ is present andis not hydrogen and wherein L¹ is present, can be prepared fromhalogenated compounds of the general formula (Y¹)(Y²)R²-L²-X that arecoupled with a protected piperidone in presence of a base, and therecovered ketone is R³-sililated, e.g. nitrilosililated, in presence ofan amine of the general formula R¹-L¹-H (L¹-H being the —NR⁴— group,with R⁴ as defined hereinabove) in acid environment to furnish thecompound of general formula (Y¹)(Y²)R²-L²-N[—CH₂—CH₂—]₂C(R³)L¹-R¹,wherein R³ is the nitrile. Thereafter, the nitrile may be hydrolyzed ina strong acid to a respective amide and further in a strong base to arespective carboxylic acid, which is reacted by peptidic methodology toa protected glycinate ester, finally deprotected to furnish the compoundof formula (I).

Certain preferred compounds of general Formula (Ic) may be preparedaccording to a Method (b) shown in Schemes 4 and 4a, starting from anaphthyl compound of general Formula F, wherein X is halogen, preferablychlorine, p is an integer having a value of 1, 2 or 3 and Y¹ and Y² areas hereinbefore defined.

Generally, compounds of general Formula H are obtained by reacting acompound of general Formula F with a piperidone, preferably4-piperidone, protected with a suitable glycol, e.g. ethylene glycol,followed by deprotection of the ketone. The reaction is carried out in apolar solvent, such as dimethyl formamide (DMF) or tetrahydrofuran(THF), in presence of a base. Suitable base may be a carbonate, e.g.potassium carbonate, sodium carbonate. The reaction may be carried outat a temperature between 0° C., and 60° C., preferably between 15° C.,and 40° C., most preferably at ambience, e.g. at room temperature. Thereaction may be kept for about 6-18 hours, preferably for about 10-14hours. The product may be purified, e.g. by chromatography, anddeprotected by heating the product under acidic conditions. Thedeprotection may be carried out in a suitable solvent, e.g. an alcohol,such as ethanol, that dissolves the acid used, e.g. hydrochloric acid.

The obtained compound of Formula H may be further reacted with asuitable substituted silane, e.g. trimethyl sililonitrile (TMSCN), in apresence of a suitable primary or secondary amine, e.g. aniline,piperidine, ethylamine, propylamine, ethylpropylamine, dipropylamine, ina suitable solvent under acidic conditions, e.g. in acetic acid,trifluoroacetic acid, benzoic acid. The reagents may be combined at atemperature lower than 60° C., preferably lower than 40° C., furtherpreferably between 0° C., and 40° C., and most preferably between 10°C., and 20° C. The reaction may be kept for about 6-18 hours, preferablyfor about 10-14 hours. After neutralizing the acid, the reaction mixturemay be extracted into an apolar solvent, such as dichloromethane, toyield the nitrile compound of formula J, which may be used withoutfurther purification.

The nitrile compounds of formula J may be further converted into thecompounds of general Formula (Ic) according to the Scheme 4a below:

The compound of the formula K is prepared by hydrolyzing the nitrilecompound of formula J in a strong acid, e.g. in concentrated sulfuricacid, nitric acid, hydrobromic acid (HBr) and hydrochloric acid (HCl).The reaction may be kept for about 6-18 hours, preferably for about10-14 hours. After neutralization of the reaction mixture, the compoundof the formula K can be purified, e.g. by reverse-phase preparativeHPLC.

The compound of the formula K may be further hydrolyzed into thecompound of formula N, e.g. with potassium hydroxide in a polar solvent,e.g. in ethylene glycol. The reagents may be combined at a temperaturebetween 110° C., and 170° C., and most preferably between 140° C., and160° C. The reaction may be kept for about 6-18 hours, preferably forabout 10-14 hours. After cooling of the reaction mixture, the compoundof the formula N can be purified, e.g. by reverse-phase preparativeHPLC. The compound of formula N may then be reacted with methylglycinate in DMF in presence of a coupling agent, e.g.1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-pyridinium-3-oxidhexafluorophosphate (known as HATU) and diisopropyl ethylamine, forabout 6-18 hours, preferably for about 10-14 hours, and purified, e.g.by reverse-phase preparative HPLC, to furnish the compound of formula Q.Additional coupling agents that may be used areN,N′-dicyclohexylcarbodiimide (DCC),3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine (EDC),3-[bis(dimethylamino)-methyliumyl]-3H-benzotriazol-1-oxidehexafluorophosphate (HBTU),1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU),1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]-methanaminiumhexafluorophosphate (COMU).

Finally, the compound of formula Q may be hydrolyzed with a base, e.g.lithium hydroxide, to release the methyl ester and furnish the crudecompound of the general Formula (Ic), e.g. in an aprotic solvent, suchas tetrahydrofuran, for about 6-18 hours, preferably for about 10-14hours. The reaction mixture may then be neutralized to pH about 7, andthen purified, e.g. by a preparative HPLC, to furnish the compound ofthe general Formula (Ic).

Compounds of general formula (Id) are prepared according to the methoddescribed for the compounds of general formulae (Ia) and (Ib).

In the reactions described hereinabove, any reactive group such as forexample an amino, alkylamino, hydroxy or carboxy group, may be protectedduring the reaction by conventional protecting groups which are cleavedafter the reaction, by methods known in the art.

The invention also relates to the stereoisomers, such as diastereomersand enantiomers, mixtures and salts, particularly the physiologicallyacceptable salts, of the compounds of general Formulae (I), (Ia), (Ib),(Ic), and (Id), and of the compounds of structural formulae 1, 2, 3, 4,5, 6, 7, 8 and 9.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), and (Id), orintermediate products in the synthesis of compounds of general Formulae(I), (Ia), (Ib), (Ic), and (Id), may be resolved into their enantiomersand/or diastereomers on the basis of their physical-chemical differencesusing methods known in the art. For example, cis/trans mixtures may beresolved into their cis and trans isomers by chromatography. Forexample, enantiomers may be separated by chromatography on chiral phasesor by recrystallisation from an optically active solvent or byenantiomer-enriched seeding.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), and (Id), andthe compounds of structural formulae 1, 2, 3, 4, 5, 6, 7, 8 and 9, maybe converted into the salts thereof, particularly physiologicallyacceptable salts for pharmaceutical use. Suitable salts of the compoundsof general Formulae (I), (Ia), (Ib), (Ic), and (Id), and of thecompounds of structural formulae 1, 2, 3, 4, 5, 6, 7, 8 and 9, may beformed with organic or inorganic acids, such as, without being limitedto hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid,lactic acid, acetic acid, succinic acid, citric acid, palmitic acid ormaleic acid. Compounds of general Formulae (I), (Ia), (Ib), (Ic) and(Id), containing a carboxy group, may be converted into the saltsthereof, particularly into physiologically acceptable salts forpharmaceutical use, with organic or inorganic bases. Suitable bases forthis purpose include, for example, sodium hydroxide, potassiumhydroxide, ammonium hydroxide, arginine or ethanolamine.

According to another aspect provided herein are uses of the compounds ofgeneral Formulae (I), (Ia), (Ib), (Ic), (Id), and (IIa), such as,without being limited to, the compounds of structural formulae 1, 2, 3,4, 5, 6, 7, 8, 9, 10 and 11, for example as oligomerization inhibitorsof Voltage-Dependent Anion Channel (VDAC), or as inhibitors ofhexokinase detachment from VDAC.

According to certain embodiments, the at least one compound is ofFormula (IIa):

wherein:

A is carbon (C);

R³ is a hydrogen, an unsubstituted or substituted amide or a heteroalkylgroup comprising 3-12 atoms apart from hydrogen atoms, wherein at leastone of said 3-12 atoms is a heteroatom, selected from nitrogen, sulfurand oxygen;

L¹ is an amino linking group —NR⁴—, wherein R⁴ is hydrogen, aC₁₋₅-alkyl, a C₁₋₅-alkylene or a substituted alkyl —CH₂R, wherein R is afunctional group selected from hydrogen, halo, haloalkyl, cyano, nitro,hydroxyl, alkyl, alkenyl, aryl, alkoxyl, aryloxyl, aralkoxyl,alkylcarbamido, arylcarbamido, amino, alkylamino, arylamino,dialkylamino, diarylamino, arylalkylamino, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl or heteroaryl;

when R³ is hydrogen, then L¹ is preferably —NH—; when R³ is heteroalkylgroup comprising 3-12 atoms, then L¹ is preferably —NC_(n)H_(2n)—, suchthat it forms a ring with R³;

R¹ is an aromatic moiety, which is optionally substituted with one ormore of C₁₋₂-alkoxy, e.g. haloalkoxy, such as C₁₋₂-perfluoroalkoxy;

L² is a linking group consisting of 4-10 atoms (apart from hydrogenatoms), optionally forming a ring, whereof at least one of the atoms isnitrogen, said nitrogen forming part of an amide group or L² is C₁₋₅alkyl or C₁₋₅ alkylene; said linking group L² bonds piperidine orpiperazine moiety at nitrogen (N) atom; preferably, L² is selected frombutanamidylene, N-methylbutanamidylene, N,N-dimethylbutanamidylene,4-hydroxybutanamidylene, 4-oxobutanamidylene,4-hydroxy-N-methylbutanamidylene, 4-oxo-N-methylbutanamidylene,2-pyrrolidonylene, pyrrolidine-2,5-dionylene,5-thioxo-2-pyrrolidinonylene and 5-methoxy-2-pyrrolidinonylene; and

R² is an aryl, optionally substituted with halogen, optionally when R²is a phenyl it is substituted with halogen, further optionally when R²is naphthyl, L² is an alkylenyl group. In a specific embodiment, R³ ishydrogen, L¹ is —NH—, and R¹ is a phenyl substituted withtrifluoromethoxy.

The invention also relates to use of the stereoisomers, enantiomers,mixtures thereof, and salts, particularly the physiologically acceptablesalts, of the compounds of general Formula (I) and (IIa).

In some embodiments, A is carbon (C), R³ is hydrogen (H), L¹ is a NHgroup, R¹ is a phenyl substituted with one trifluoromethoxy, L² ispyrrolidine-2,5-dione, and R² is a phenyl substituted with a chlorine atthe para position.

In some embodiments, A is carbon (C), R³ is a C(O)NCH₂C(O)OH group andis connected to both A and L¹, L¹ is a NCH₂ group and is connected toboth R¹ and R³, R¹ is a phenyl, L² is methylene C¹ alkylene and R² is anaphthyl.

According to certain exemplary embodiments, methods of the presentinvention comprise administering to the subject compounds according tothe general Formula (IIa), having the structural Formulae 10 and 11:

The compound of Formula 10 is also identified herein as AKOS022 orAKOS022075291.

The compound of Formula 11 is also identified herein as DIV 00781.

The compounds of general Formula (IIa) such as, without being limitedto, the compounds of structural formulae 10 and 11, may be convertedinto the salts thereof, particularly physiologically acceptable saltsfor pharmaceutical use. Suitable salts of the compounds of generalFormulae (IIa), such as, without being limited to, the compounds ofstructural formulae 10 and 11, may be formed with organic or inorganicacids, such as, without being limited to hydrochloric acid, hydrobromicacid, sulphuric acid, phosphoric acid, lactic acid, acetic acid,succinic acid, citric acid, palmitic acid or maleic acid. Compounds ofgeneral Formula (IIa) containing a carboxy group, may be converted intothe salts thereof, particularly into physiologically acceptable saltsfor pharmaceutical use, with organic or inorganic bases. Suitable basesfor this purpose include, for example, sodium salts, potassium salts,arginine salts, ammonium salts, or ethanolamine salts.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), and(IIa), particularly the specific compounds of Formulae 1, 2, 3, 10 and11, are inhibitors of Voltage-Dependent Anion Channel (VDAC)oligomerization and apoptosis. The effect of the compounds of theinvention and of the specific compounds of formulae 1, 2, 3, 10 and 11on VDAC oligomerization, i.e. their ability to inhibit VDAColigomerization, may be determined, e.g. by Bioluminescence ResonanceEnergy Transfer (BRET2) technology that allows to directly monitor theoligomeric state of VDAC molecules in the native membrane in cells inlive. BRET2 screening may be carried out as described in the art (e.g.Keinan et al., 2010. Mol Cell Biol 30, 5698-5709).

The compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), and(IIa), particularly the compound having Formula 1 (VBIT-4), inhibit thedetachment of hexokinase (HK), particularly HK-I, from the mitochondrialVDAC. Without wishing to be bound by any theory or mechanism of action,the ability of the compounds of the invention, particularly VIBIT-4, toinhibit detachment of HK from VDAC contributes to their therapeuticeffect on CNS-associated diseases, including mood disorders, psychoticdisorders (particularly schizophrenia), Alzheimer disease, Parkinsondisease, known to be associated with impaired brain energy metabolismdue to HK detachment. Impaired mitochondrial energy metabolism was alsoshown to be involved in amyotrophic lateral sclerosis (ALS).

According to certain embodiments, the CNS-associated disease that can betreated with the compound of the present invention is selected from thegroup consisting of psychotic disorders, mood disorders andneurodegenerative disease. Each possibility represents a separateembodiment of the present invention.

According to some embodiments, the psychotic disorder is selected fromthe group consisting of, but not limited to, schizophrenia, autismspectrum disorder and anorexia nervosa. Each possibility represents aseparate embodiment of the present invention.

According to certain exemplary embodiments, the psychotic disorder isschizophrenia.

According to certain embodiments, the mood disorder is selected from thegroup consisting of, but not limited to, bipolar disorder, majordepressive disorder, persistent depressive disorder (also known asdythemia) and anxiety disorder. Each possibility represents a separateembodiment of the present invention.

According to certain exemplary embodiments, the mood disorder is bipolardisorder.

According to certain embodiments, the neurodegenerative disease isselected from the group consisting of, but not limited to, Alzheimer'sdisease, Parkinson disease and Amyotrophic Lateral Sclerosis (ALS). Eachpossibility represents a separate embodiment of the present invention.

According to certain exemplary embodiments, the neurodegenerativedisease is Alzheimer's disease.

According to certain additional or alternative embodiments, theneurodegenerative disease is Parkinson disease.

According to certain additional or alternative embodiments, theneurodegenerative disease is ALS.

As exemplified herein, an exemplary compound of the invention, VBIT-4(having formula 1) inhibits mitochondrion-bound HK detachment (FIG. 13).In addition to the metabolic function assigned to mitochondrion-bound HK(HK-I and HK-II), namely the coupling of cytosolic glycolysis tomitochondrial oxidative phosphorylation, it was shown that VDAC1-boundHK also prevents the release of pro-apoptotic factors and subsequentapoptosis accompanied with detachment of HK (see e.g. Shoshan-Barmatz Vet al. 2009. Biophys Acta 1787, 421-430; Arzoine, L et al. 2009. J.Biol. Chem. 284, 3946-3955). Several pro-apoptotic agents have beenshown to induce VDAC1-HK complex dissociation (see, e.g. Shoshan-BarmatzV et al. 2015. Biochim. Biophys. Acta 1848, 2547-2575). The presentinvention now shows that VBIT-4 inhibited HK detachment, as induced byapoptosis induction. This finding supports the concept of HK detachmentbeing a pre-requisite for apoptosis induction. VBIT-4 inhibition of HKdetachment suggests that such detachment is associated with VDAC1oligomerization, with VBIT-4 inhibiting oligomerization and preventingHK detachment. In addition, attachment of HK-I and/or HK-II tomitochondrial VDAC has been was shown play a significant role in properenergy metabolism of mammalian cell, particularly of neuronal cells.Without wishing to be bound by certain specific mechanism or mode ofaction, the inhibiting activity of the compounds of the presentinvention on HK detachment contribute to its therapeutic effect on CNSdisorders, particularly psychotic disorders, mood disorders and theneurodegenerative disease Alzheimer's disease, Parkinson disease andALS.

The compounds of the invention exemplified by the compound of Formula 1(VBIT-4) were found not toxic and well tolerated by rats. No mortalityand no treatment-related clinical signs were observed during a studywith 40 mg/kg administered via oral gavage (data not shown). Both foodintake and body weight were normal in all animals. In addition, nosignificant changes in hematology and serum chemistry were observed.Post mortem examination showed no significant changes in organ weight,or macroscopic examination of liver, lung, testis, tongue, marrow boneand pathological tissues and no abnormal clinical symptoms wereobserved.

VBIT-4 showed an elimination half-life (PK) of 7.6 h, a stable metabolicprofile and moderate intestinal permeability. VBIT-4 showed high plasmaprotein binding, with the bound compound fraction possibly serving as areservoir from which slow release occurs (data not shown).

Animal models may serve as a resource for developing and evaluatingtreatments for disease associated with CNS disorders.

Features that characterize schizophrenia in animal models typicallyextend to schizophrenia in humans. Thus, efficacy in such animal modelsis expected to be predictive of efficacy in humans. Other psychiatricdiseases including schizotypical and schizoaffective disorder, otheracute- and chronic psychoses, and bipolar disorder (in particular,mania), have an overlapping symptomatology with schizophrenia. Variousanimal models of schizophrenia are known in the art.

One animal model of schizophrenia is protracted treatment withmethionine. Methionine-treated mice exhibit deficient expression ofGAD67 in frontal cortex and hippocampus, similar to those reported inthe brain of postmortem schizophrenia patients. They also exhibitprepulse inhibition of startle and social interaction deficits(Tremonlizzo et al., 2002. PNAS, 99, 17095-17100). Another animal modelof schizophrenia is methylazoxymethanol acetate (MAM)-treatment in rats.Pregnant female rats are administered MAM (20 mg/kg, intraperitoneal) ongestational day 17. MAM-treatment recapitulates a pathodevelopmentalprocess to schizophrenia-like phenotypes in the offspring, includinganatomical changes, behavioral deficits and altered neuronal informationprocessing. More specifically, MAM-treated rats display a decreaseddensity of parvalbumin-positive GABAergic interneurons in portions ofthe prefrontal cortex and hippocampus. In behavioral tests, MAM-treatedrats display reduced latent inhibition. Latent inhibition is abehavioral phenomenon where there is reduced learning about a stimulusto which there has been prior exposure with any consequence. Thistendency to disregard previously benign stimuli, and reduce theformation of association with such stimuli is believed to preventsensory overload. Low latent inhibition is indicative of psychosis.Latent inhibition may be tested in rats in the following manner. Ratsare divided into two groups. One group is pre-exposed to a tone overmultiple trials. The other group has no tone presentation. Both groupsare then exposed to an auditory fear conditioning procedure, in whichthe same tone is presented concurrently with a noxious stimulus, e.g. anelectric shock to the foot. Subsequently, both groups are presented withthe tone, and the rats' change in locomotor activity during tonepresentation is monitored. After the fear conditioning the rats respondto the tone presentation by strongly reducing locomotor activity.However, the group that has been exposed to the tone before theconditioning period displays robust latent inhibition: the suppressionof locomotor activity in response to tone presentation is reduced.MAM-treated rats, by contrast show impaired latent inhibition. That is,exposure to the tone previous to the fear conditioning procedure has nosignificant effect in suppressing the fear conditioning. Amphetamines,while known to be used for treating psychiatric disorders, may inducesymptoms of psychosis very similar to those of acute schizophreniaspectrum psychosis. Amphetamine is known to disrupt latent inhibition,and is thus used as a model for primary psychotic disorders includingschizophrenia.

Apomorphine-induced climbing (AIC) and Apomorphine-induced stereotype(AIS) in mice is another animal model that may be useful in thisinvention. The compounds of the invention are administered to mice at adesired dose level (e.g., via intraperitoneal administering).Subsequently, e.g., thirty minutes later, experimental mice arechallenges with apomorphine (e.g., with 1 mg/kg sc). Five minutes afterthe apomorphine injection, the sniffing-licking-gnawing syndrome(stereotyped behavior) and climbing behavior induced by apomorphine arescored and recorded for each animal. Readings can be repeated every 5min during a 30-min test session. Scores for each animal are totaledover the 30-min test session for each syndrome (stereotyped behavior andclimbing). If an effect reach at least of 50% inhibition, an ID₅₀ value(95% confidence interval) is calculated using a nonlinear least squarescalculation with inverse prediction. Mean climbing and stereotype scorescan be expressed as a percent of control values observed in vehicletreated mice that receive apomorphine only (see Grauer S M et al. 2009.Psychopharmacology 204, 37-48).

In another well-established preclinical model of schizophrenia, ratsexposed chronically to ketamine, an uncompetitive N-methyl-D-aspartate(NMDA) receptor antagonist, produces positive and negative psychoticsymptoms and cognitive impairment. Long-Evans male rats are injectedintraperitoneally with ketamine (0.30 mg/kg, twice a day) for two weeksduring adolescence (2 month-old). Rats are behaviorally tested when theyreach adulthood (approximately 4-5 month-old) for the behavioralsymptoms to ketamine exposure and for the efficacy of treatment with thecompounds of the invention to alleviate those symptoms (see, e.g.,Enomoto et al. 2003. Progress in Neuro-Psychopharmacology & BiologicalPsychiatry 33, 668-675).

Such animal models of schizophrenia may be used to assay theeffectiveness of the methods and compositions of the invention intreating schizophrenia or bipolar disorder (in particular, mania).

Depression is a mood disorder that can be classified per se or can beassociated with other psychiatric states or with general medicalconditions, typically chronic and/or life threatening diseases,including, for example, cancer, HIV/AIDS, ALS and multiple sclerosis.The efficacy of the methods and compositions of the present invention intreating depression may be assessed in animal models of depression. Anexemplary animal model of depression is described in Example 8hereinbelow.

Alzheimer disease (AD), which is the most common form of dementia, ischaracterized by gradual cognitive decline, eventually leading to death.Worldwide, 36 million people older than 65 years live with dementia, andthis number is anticipated to double by 2030 and reach 115 million by2050. The disease is characterized by the occurrence of brain senileplaques and neurofibrillary tangles, and is associated with the loss ofbrain synapses and synaptic dysfunction, inflammatory responses, andmitochondrial structural and functional abnormalities. The senileplaques associated with the disease contain a 39-43 amino acid-longamyloid-β peptide (Aβ), a fragment of the amyloid protein precursor,APP. Over-production of Aβ represents one of the pathological hallmarksof AD; it was shown that different aggregated forms of the Aβ stimulatereactive oxygen species (ROS) production in neurons. It has beenrecently shown that Aβ triggers neuronal oxidative stress by interferingwith mitochondrial HKI activity and subcellular localization.

Dementia and cognitive impairments are hallmarks of Alzheimer's disease,and various animal models of these features are known in the art. Theeffect of exemplary product of the invention (VBIT-4, having formula 1)on AD on 5XFAD transgenic mice with AD-like disease is described inExample 6 hereinbelow.

Parkinson's disease (PD) is a neurological disorder characterized by adecrease of voluntary movements. The afflicted patient has reduction ofmotor activity and slower voluntary movements compared to the normalindividual. The patient has characteristic “mask” face, a tendency tohurry while walking, bent over posture and generalized weakness of themuscles. There is a typical “lead-pipe” rigidity of passive movements.Another important feature of the disease is the tremor of theextremities occurring at rest and decreasing during movements. Theetiology of PD is unknown. It belongs to a group of the most commonmovement disorders named parkinsonism, which affects approximately oneperson per one thousand. These other disorders grouped under the name ofparkinsonism may result from viral infection, syphilis, arteriosclerosisand trauma and exposure to toxic chemicals and narcotics. Nonetheless,it is believed that the inappropriate loss of synaptic stability maylead to the disruption of neuronal circuits and to brain diseases.Whether as the result of genetics, drug use, the aging process, viralinfections, or other various causes, dysfunction in neuronalcommunication is considered the underlying cause for many neurologicdiseases, such as PD (Myrrhe van Spronsen and Casper C. 2010.Hoogenraad, Curr. Neurol. Neurosci. Rep., 10, 207-214).

Regardless of the cause of the disease, the main pathologic feature isdegeneration of dopaminergic cells in basal ganglia, especially insubstantia nigra. Due to premature death of the dopamine containingneurons in substantia nigra, the largest structure of the basal ganglia,the striatum, will have reduced input from substantia nigra resulting indecreased dopamine release. The understanding of the underlyingpathology led to the introduction of the first successful treatmentwhich can alleviate Parkinson's disease. Virtually all approaches to thetherapy of the disease are based on dopamine replacement. Drugscurrently used in the treatment can be converted into dopamine aftercrossing the blood brain barrier, or they can boost the synthesis ofdopamine and reduce its breakdown. Unfortunately, the main pathologicevent, degeneration of the cells in substantia nigra, is not helped. Thedisease continues to progress and frequently after a certain length oftime, dopamine replacement treatment will lose its effectiveness.

There are a number of animal models for PD. Exemplary animal models forPD include the reserpine model, the methamphetamine model, the6-hydroxydopamine (6-OHDA) model, the1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model, the paraquat(PQ)-Maneb model, the rotenone model, the 3-nitrotyrosine model andgenetic models using transgenic mice. Transgenic models include micethat over express α-synuclein, express human mutant forms ofα-synuclein, or mice that express LRKK2 mutations (see, e. g. Ranjita Bet al. 2002. BioEssays 24, 308-318, a review).

Amyotrophic Lateral Sclerosis (ALS) is characterized by degeneration ofmotor neurons and also by neuronal degeneration in the entorhinal cortexand hippocampus, memory deficits, and neuronal hyperexcitability indifferent brain areas such as the cortex. Animal models for ALS areknown in the art. For example, rats expressing human Cu—Zn superoxidedismutase (SOD1) mutations are used as model animals for assessing theeffect of various drugs on ALS. These rats develop a motor syndrome withsymptoms and pathological features of the human disease (Howland D S etal. 2002. Proc Natl Acid Sci USA 99, 1604-1609).

The VDAC inhibitory piperazine and/or piperidine derivatives of thepresent invention can be administered alone, or within a pharmaceuticalcomposition containing the VDAC inhibitory compounds of the inventiontogether with a pharmaceutically acceptable carrier or excipient.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), and(IIa), such as, without being limited to, the compounds of structuralformulae 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, particularly the specificcompounds of Formulae 1, 2, 3, 10 and 11, and the pharmaceuticallyacceptable salts thereof, may be formulated in a pharmaceuticalcomposition, optionally comprising other active substances, and one ormore of inert conventional excipients, as known to the skilled artisans.The pharmaceutical compositions may be prepared according to the generalguidance provided in the art, e.g. by Remington, The Science andPractice of Pharmacy (formerly known as Remington's PharmaceuticalSciences). ISBN 978-0-85711-062-6. The pharmaceutical compositions, e.g.in the form of solid dosage forms, topical dosage form, and/orparenteral dosage forms, e.g. tablets, capsules, creams, ointments,patches, injections, and others as known in the art constitute anotheraspect of the invention.

Particularly, the compounds of general Formulae (I), (Ia), (Ib), (Ic),(Id), and (IIa), particularly of structural formulae 1, 2, 3, 4, 5, 6,7, 8, 9, 10 and 11, more particularly the specific compounds of Formulae1, 2, 3, 10 and 11, and the pharmaceutically acceptable salts thereof,may be formulated as nanoparticles. The nanoparticles may be prepared inwell-known polymers, e.g. polylactic-co-glycolic acid, e.g. as describedin H. K. Makadia, S. J. Siegel, Poly Lactic-co-Glycolic Acid (PLGA) asBiodegradable Controlled Drug Delivery Carrier, Polymers (Basel), 3(2011) 1377-1397; and others. Generally, the compounds may beco-dissolved with the polymer in a suitable organic solvent, and theorganic phase may be then dispersed in an aqueous phase comprisingstabilizers and/or surface active agents. The stabilizers may be, e.g.polyvinyl alcohol, with molecular weights from about 89000 to 98000, andhydrolysis degree from about 99%. Upon evaporation of organic solventfrom the aqueous phase, the nanoparticles may be purified, e.g. bycentrifugation and washing.

The encapsulated compounds, e.g. in form of nanoparticles, of generalFormulae (I), (Ia), (Ib), (Ic), (Id), and (IIa), such as, without beinglimited to, the compounds of structural formulae 1, 2, 3, 4, 5, 6, 7, 8,9, 10 and 11, particularly the specific compounds of Formulae 1, 2, 3,10 and 11, and the pharmaceutically acceptable salts thereof, may beadvantageously used in various routes of administering. Intranasal routemay be suitable mode of administering in this regard. Alternatively, thenanoparticles may be administered systemically to accumulate incancerous tissues.

The dose of compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id),and (IIa), such as, without being limited to, the compounds ofstructural formulae 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, particularlythe specific compounds of Formulae 1, 2, 3, 10 and 11, and thepharmaceutically acceptable salts thereof, required to achieve treatmentor prevention of a disease or a disorder or a condition usually dependson the pharmacokinetic and pharmacodynamic properties of the compoundwhich is to be administered, the patient, the nature of the disease,disorder or condition and the method and frequency of administering.Suitable dosage ranges for compounds of general Formulae (I), (Ia),(Ib), (Ic), (Id), and (IIa), such as, without being limited to, thecompounds of structural formulae 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11,particularly the specific compounds of Formulae 1, 2, 3, 10 and 11, andthe pharmaceutically acceptable salts thereof, may be from 1.0 to 100mg/kg body weight.

EXAMPLES Materials

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), cytochalasin B,dimethyl sulfoxide (DMSO), DL-dithiothreitol (DTT), EDTA, HEPES,leupeptine, phenylmethylsulfonyl fluoride (PMSF), and Tris werepurchased from Sigma (St. Louis, Mo.). Coelenterazine (Deep Blue C[DBC]) was obtained from Bioline (Taunton, Mass.). Digitonin came fromCalbiochem-Novobiochem (Nottingham, UK). Rabbit monoclonal antibodiesagainst VDAC1 (ab154856) and mouse monoclonal antibodies against GAPDH(ab9484) came from Abeam (Cambridge, UK). Monoclonal antibodies againstactin were obtained from Millipore (Billerica, Mass.) Horseradishperoxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies wereobtained from Promega (Madison, Wis.). Dulbecco's modified Eagle'smedium (DMEM) and the supplements fetal bovine serum (FBS), L-glutamineand penicillin-streptomycin were purchased from Biological Industries(Beit-Haemek, Israel). The compound of Formula 10 (AKOS022) was obtainedfrom AKosConsulting & Solutions GmbH (Germany), under catalogue numberAKOS022075291.

Methods LC-MS Analysis

The chromatography was performed using a regular Bridge C18 column4.6×50 mm, 3.5 μm, kept at 40° C. The materials were eluted at 2 mL/min,with mixture of 0.01M aqueous solution of ammonium carbonate andacetonitrile, with acetonitrile ramping from 5% to 100% for periods asdescribed below and eluting with 100% acetonitrile, with detection atthe target mass.

Tissue Culture

HEK-293, HeLa, SH-SY5Y and K-Ras-transformed Bax^(−/−)/Bak^(−/−) mouseembryonic fibroblast (MEF) cell lines were grown at 37° C. under anatmosphere of 95% air and 5% CO₂ in DMEM supplemented with 10% FBS, 2 mML-glutamine, 1000 U/ml penicillin and 1 mg/ml streptomycin. T-REx-293cells (HEK cells stably containing the pcDNA6/TR regulatory vector andthus expressing the tetracycline repressor; Invitrogen) stablyexpressing hVDAC1-shRNA and showing low (10-20%) endogenous VDAC1expression (referred to herein as T-REx-pS10) were grown under the sameconditions as HEK-293 cells, with an addition of 5 μg/ml blasticidin.

Hexokinase Detachment from Mitochondria

Cells treated with apoptosis inducers in the absence or presence of thetested compounds were harvested, washed twice with PBS, pH 7.4 andgently resuspended at 6 mg/ml in ice-cold buffer (100 mM KCl, 2.5 mMMgCl₂, 250 mM sucrose, 20 mM HEPES/KOH pH 7.5, 0.2 mM EDTA, 1 mMdithiothreitol, 1 μg/ml leupeptin, 5 mg/ml cytochalasin B and 0.1 mMPMSF) containing 0.025% digitonin and incubated for 10 min on ice.Samples were centrifuged at 10,000×g (relative centrifugal force—RCF) at4° C. for 5 min to obtain supernatants (cytosolic extracts free ofmitochondria) and pellet (fraction that contains mitochondria).Hexokinase I released to the cytosol was analyzed by immunoblottingusing anti-hexokinase-I-specific antibodies. Anti-VDAC1 and anti-GAPDHantibodies were used to verify that the cytosolic extracts aremitochondria-free.

Preparation of Intermediate 1

Step A

Intermediate 1 was synthesized according to the scheme below.

The starting material reagent 1 (p-trifluoromethoxy-bromobenzene;1-Bromo-4-(trifluoromethoxy)benzene) was used. To a solution of reagent1 (2.41 g, 10 mmol) in toluene (50 mL) were consecutively added thefollowing compounds: reagent 2 (1-Boc-piperazine; tert-butylpiperazine-1-carboxylate) (1.68 g, 9 mmol), Pd₂(dba)₃(tris-(dibenzylidenacetone)dipalladium) (290 mg, 0.5 mmol),2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (311 mg, 0.5 mmol)and sodium t-butoxide (1.92 g, 20 mmol). The mixture was refluxed underN₂ atmosphere overnight. The solvent was evaporated to provide crudereagent 3 (tert-butyl4-(4-(trifluoromethyl)-phenyl)-piperazine-1-carboxylate) as a residue.

Step B

Reagent 3 was directly used for next step without further purification.

Boc group was removed by acid hydrolysis according to the scheme below

The mixture comprising crude reagent 3 in 50 mL of concentratedhydrochloric acid and 50 mL of dichloromethane was stirred for 1.5 hoursat room temperature. After phase separation, the dichloromethane phasewas discarded, and the aqueous phase was evaporated in vacuo to dryness.The residue was dissolved in 50 mL of aqueous sodium hydroxide solution,2.0 M, and 50 mL dichloromethane were added and stirred for additional1.5 hours. The organic phase was collected and concentrated in vacuo toprovide Intermediate 1 as brown oil (2.0 g, 80% yield for steps A andB).

The Intermediate 1 was analyzed using LC-MS method as described abovewith ramping over 1.6 minutes and elution for 1.4 minutes, withdetection at +246. A representative chromatogram and respective massspectra of two peaks of interest relating to Intermediate 1 arerepresented in FIG. 1.

Example 1: Preparation of Compound of Formula 2 (VBIT-3)

The compound of Formula 2(1-(4-chlorophenyl)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)pyrrolidine-2,5-dione;VBIT-3) was synthesized according to the reaction scheme below:

To a solution of Intermediate 1 (207 mg, 1 mmol) in methanol (2 mL) wasadded reagent 5 (1-(4-Chlorophenyl)-1H-pyrrole-2,5-dione) (246 mg, 1mmol). The reaction was stirred at room temperature overnight. The finalmixture was concentrated and purified by preparative reverse-phase HPLCto provide compound of Formula 2 (VBIT-3) as white solid (100 mg, 22%yield).

The product (compound of Formula 2 (VBIT-3)) was analyzed using LC-MSmethod as described above with ramping over 3 minutes and elution for 1minute, with detection at +453. The chromatogram is represented in FIG.2 a.

The NMR spectra were obtained on 400 MHz apparatus (by Varian).

¹H NMR (400 MHz, DMSO-d₆): δ7.592 (d, J=2.2 Hz, 2H), 7.339 (d, J=2.2 Hz,2H), 7.201 (d, J=2.2 Hz, 2H), 7.021 (d, J=2.1 Hz, 2H), 4.137 (dd, J=1.3Hz, 1H), 3.159 (m, J=1.1 Hz, 4H), 3.00 (m, J=2.4 Hz, 3H), 2.871 (m,J=1.3 Hz, H), 2.680 (m, J=2.0 Hz, 2H). The spectrum is shown in the FIG.2 b.

Example 2 Preparation of Compound of Formula 1 (VBIT-4

The compound of Formula 1(N-(4-chlorophenyl)-4-hydroxy-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanamide; VBIT-4) was synthesized according to thereaction scheme below:

Step A

A mixture of Intermediate 1 (2.0 g, 8 mmol) and furan-2(5H)-one(2(5H)-Furanone) (1.3 g, 16 mmol) in methanol (MeOH) (5 mL) was stirredat room temperature overnight. The mixture was evaporated in vacuo andthe residue was purified by reverse phase preparative HPLC to provideIntermediate 2[3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-dihydrofuran-2(3H)-one)as white solid (1.3 g, 0.4 mmol, 50% yield).

The product (Intermediate 2) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minutes, with detection at +330. The chromatogram is represented in FIG.3.

Step B

To a solution of 4-chloroaniline (254 mg, 2 mmol) in toluene (5 mL)trimethyl aluminum (AlMe₃) was added (2.0 M in toluene, 2 mL). Afterstirring for 10 minutes, Intermediate 2 (330 mg, 1.0 mmol) was added tothe solution and the resulting mixture was heated to 80° C. for 8 hours.After cooling to room temperature, the solvent was evaporated in vacuoand the residue was purified by reverse preparative HPLC to afford thecompound of Formula 1 (VBIT-4) as white solid (200 mg, 44% yield).

The product (Compound of Formula 1) was analyzed using LC-MS method asdescribed above with ramping over 3 minutes and elution for 1 minute,with detection at +457. The chromatogram is represented in FIG. 4 a.

The NMR spectra were obtained on 400 MHz apparatus (by Varian).

¹H NMR (400 MHz, DMSO-d₆): δ10.081 (s, H), 7.601 (d, J=0.5 Hz, 2H),7.341 (d, J=1.2 Hz, 2H), 7.177 (d, J=2.2 Hz, 2H), 6.980 (d, J=2.3 Hz,2H), 4.538 (dd, J=1.2 Hz, 1H), 3.561 (m, J=1.3 Hz, H), 3.440 (m, J=1.4Hz, H), 3.112 (m, J=1.2 Hz, 5H), 2.807 (m, J=1.6 Hz, 2H), 2.709 (m,J=1.5, Hz, 2H), 2.400 (m, J=1.4, Hz, H), 2.150 (m, J=1.4, Hz, H). Thespectrum is shown in the FIG. 4 b.

Example 3: Preparation of Compound of Formula 3 (VBIT-12)

Preparation of compound of Formula 3(2-(1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carboxamido)aceticacid; VBIT-12):

Step 1

1-(Chloromethyl)naphthalene (8.8 g, 50 mmol) was dissolved indimethylformamide (DMF) (100 mL), and potassium carbonate (13.8 g, 100mmol) was added, followed by 4-piperidone ethylene ketal(1,4-dioxa-8-azaspiro[4.5]decane) (7.2 g, 50 mmol). The mixture wasstirred at room temperature overnight. The solvent was evaporated invacuo and the residue was purified by chromatography in silica gel(eluting with dichloromethane) to provide pure naphthylated ketal(Intermediate 3) as white solid (8.5 g, 60% yield).

Intermediate 3 was analyzed using LC-MS method as described above withramping over 1.6 minutes and elution for 1.4 minute, with detection at+283. The chromatogram is represented in FIG. 5.

Step 2

A solution of Intermediate 3 (product of Step 1) (1.42 g, 5 mmol) in 20ml of 3N hydrochloric acid (HCl) in ethanol (EtOH) was refluxedovernight. The resulting mixture was concentrated in vacuo to provideIntermediate 4 (1-(naphthalen-1-ylmethyl)piperidin-4-one), which wasused with no further purification.

The product (Intermediate 4) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +239. The chromatogram is represented in FIG.6.

Step 3

Intermediate 4 (N-methylnaphthyl-4-piperidinone) (2.4 g, 10 mmol) andaniline (930 mg, 10 mmol) were dissolved in glacial acetic acid (AcOH)(25 mL). Thereafter, trimethylsilyl cyanide (TMSCN) was added dropwise(1.3 mL, 10 mmol) over a 10-min period, maintaining the temperaturebelow 40° C. using a cold water bath. The solution was stirred overnightand then poured into ammonium hydroxide ice mixture, formed by 50 mL ofconcentrated ammonium hydroxide solution and 100 g of crushed ice.Additional concentrated ammonium hydroxide was slowly added until pHrose to 10. The resultant mixture was extracted three times with 100 mLof chloroform, and the combined organic layers were dried over sodiumsulfate, filtered and concentrated to a yellow nitrile residue(Intermediate 5,1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carbonitrile)which was used in the next step directly without further purification.

The product (Intermediate 5) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +341. The chromatogram is represented in FIG.7.

Step 4

The nitrile (Intermediate 5) was hydrolyzed according to the schemebelow:

Intermediate 5 (the product of step 3) was mixed with 10 mL ofconcentrated sulfuric acid (H₂SO₄). The mixture was stirred at roomtemperature overnight. A concentrated ammonium hydroxide solution wasslowly added until pH rose to 10. The final mixture was concentrated andpurified by reverse phase preparative HPLC to provide the amide(Intermediate 6,1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carboxamide) aswhite solid (400 mg, 11% yield for the steps 2-4).

The product (Intermediate 6) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +359. The chromatogram is represented in FIG.8.

Step 5

Intermediate 6 was further hydrolyzed to carboxylic acid according tothe scheme below:

Intermediate 6 (360 mg, 1.0 mmol) was dissolved in ethylene glycol (10mL), and potassium hydroxide (KOH) (280 mg, 5 mmol) was added. Theresulting mixture was heated to 150° C., and stirred overnight. Aftercooling to room temperature, the final mixture was concentrated in vacuoand purified by reverse phase preparative HPLC to provide the freecarboxylic acid (Intermediate 7,1-(naphthalen-1-ylmethyl)-4-(phenylamino)-piperidine-4-carboxylic acid)as white solid (200 mg, 50% yield).

The product (Intermediate 7) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +360. The chromatogram is represented in FIG.9.

Step 6

Intermediate 7 was glycinated with methyl 2-aminoacetate (methylglycinate) according to the scheme below:

Intermediate 7 (180 mg, 0.5 mmol), HATU(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]-pyridinium3-oxid hexafluorophosphate) (380 mg, 1.0 mmol),N,N-diisopropylethylamine (DIEA) (260 mg, 2.0 mmol), and methylglycinate (90 mg, 1.0 mmol) were dissolved in dimethyl formamide (DMF)(10 mL), and the solution was stirred at room temperature overnight. Theresulting mixture was concentrated in vacuo and purified by reversephase preparative HPLC to provide the glycinate methyl ester(Intermediate 8, methyl(1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carbonyl)glycinate)as white solid (100 mg, 46% yield).

The product (Intermediate 8) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +431. The chromatogram is represented in FIG.10.

Step 7

Intermediate 8 (the glycinate methyl ester product of Step 6) washydrolyzed with lithium hydroxide in tetrahydrofuran, according to thescheme below:

To a solution of Intermediate 8 (100 mg, 0.23 mmol) in 5 mL of THF, asolution of lithium hydroxide (LiOH) (40 mg, 1.0 mmol) in 5 mL of waterwas added, the resulting mixture was stirred at room temperatureovernight. Thereafter, the pH was adjusted to about 7 with 1.0 N HCl.The mixture was concentrated in vacuo and purified by preparative HPLCto provide the compound of Formula 3 (VBIT-12) (20 mg, 20% yield) aswhite solid.

The product (compound of Formula 3; IUPAC name:2-(1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carboxamido)aceticacid) was analyzed using LC-MS method as described above with rampingover 3 minutes and elution for 1 minute, with detection at +417. Thechromatogram is represented in FIG. 11 a.

The NMR spectra were obtained on 400 MHz apparatus (by Varian).

¹H NMR (400 MHz. DMSO/D2O-d6): δ8.63 (d, H), 8.1 (s, H), 7.9 (d, J=1.2Hz, 2H), 7.89 (d, J=2.2 Hz, 2H), 7.87 (d, J=2.3 Hz, 2H), 7.71 (dd, J=1.2Hz, 2H), 7.67 (d, J=1.3 Hz, 2H), 7.16 (2, J=1.4 Hz, 2H), 6.78 (m, 4H),3.80 (s, 2H), 3.71 (s, 2H), 2.31 (d, J=2.4, Hz, 2H), 2.25 (s, 2H), 2.17(t, J=2.4, 2H), 1.98 (t, J=1.9, 2H), 1.88 (t, J=1.8, 2H)

The spectra in d6-DMSO and in d6-DMSO with D₂O are shown in the FIGS.11b and 11c respectively.

Example 4: Chiral Separation of Compound of Formula 1 (VBIT-4)Enantiomers

Racemic compound of Formula 1 (VBIT-4) was analyzed by analytical chiralHPLC. Briefly, the material was eluted on Chiralpak-IC3 column (4.6×100mm, 3 μm), kept at 35° C., at 2 mL/min, with acetonitrile and 20% of0.1% solution of DEA in methanol. Two peaks, with 0.38 min difference inretention time (2.32 and 2.7 min), were obtained in expected ratio asabout 50.0%. Preparative chiral HPLC was then conducted. Each peak wascollected separately. The enantiomers were analyzed by 400 MHz NMR butwere not discernable in deuterated DMSO.

¹H NMR (400 MHz, DMSO-d₆): δ10.081 (s, H), 7.601 (d, J=0.5 Hz, 2H),7.341 (d, J=1.2 Hz, 2H), 7.177 (d, J=2.2 Hz, 2H), 6.980 (d, J=2.3 Hz,2H), 4.538 (dd, J=1.2 Hz, 1H), 3.561 (m, J=1.3 Hz, H), 3.440 (m, J=1.4Hz, H), 3.112 (m, J=1.2 Hz, 5H), 2.807 (m, J=1.6 Hz, 2H), 2.709 (m,J=1.5, Hz, 2H), 2.400 (m, J=1.4, Hz, H), 2.150 (m, J=1.4, Hz, H).

FIG. 12a demonstrates a representative NMR spectrum in deuterated DMSOrelating to the separated single enantiomer of the compound of Formula1, VBIT-4-1 (also referred to as BGD-4-1). FIG. 12b demonstrates arepresentative NMR spectrum in deuterated DMSO relating to the separatedsingle enantiomer of the compound of Formula 1, VBIT-4-2 (also referredto as BGD-4-2).

Example 5: Effect of Compound of Formula 1 on HK-I Release fromMitochondria

HEK-293 cells were incubated without and with the racemic compound ofFormula 1 (VBIT-4) (10 or 15 μM) for 2 hours and then with or withoutselenite (15 μM, 4 h), trypsinized, washed with PBS, proteinconcentration was determined and harvested.

Hexokinase-I (HK-I) release from the mitochondria was determined asdescribed above in the Methods section (hexokinase detachment frommitochondria). Briefly, to assess HK-I release, cells were incubated onice for 10 min with 0.025% digitonin, centrifuged, and the pellet(mitochondria—Mito) and supernatants (cytosol—Cytos) were subjected toSDS-PAGE and immunoblotting, using anti-HK-I, antibodies. Anti-VDAC1 andanti-GAPDH antibodies were used to verify that the cytosolic extractsare mitochondria-free. The results of HK release from the mitochondriaas induced by selenite are presented as immunoblots, with the cytosolicand mitochondrial fractions confirmed by immunoblotting of GAPDH(glyceraldehyde-3-phosphate dehydrogenase) and VDAC1, respectively, inFIG. 13. The quantitative data of selenite-induced HK-I release to thecytosol by VBIT-4 is presented as relative units (RU).

The results show that apoptosis induction by selenite resulted in thedetachment of mitochondria-bound HK that was subsequently detected inthe supernatant of digitonin-treated cells. This selenite-induced HKdetachment was inhibited in the presence of VBIT-4. The results showthat in the absence of apoptosis induction, most of the HK is bound tothe mitochondria, indicating that VBIT-4 inhibited HK detachment, asinduced by apoptosis induction.

Example 6: Effect of Compound of Formula 1 on Learning and Memory Taskof 5XFAD Transgenic Mice with AD-Like Disease

The effect of the compound of Formula 1 (VBIT-4) on learning and memorytask of 5XFAD transgenic mice with AD-like disease (Webster, S. J., etal, 2014. Frontiers in genetics 5, 88, ([5XFAD B6.Cg-Tg APPSwFlLon,PSEN1*M146Ln*L286V6799Vas/J]) was tested using the radial-arm water mazefor testing learning and memory task. These mice present detectablephenotypes of intracellular and extracellular amyloid plaques at 2months of age; develop cognitive impairments at 4-5 months and exhibitsneuronal death at 9 months.

VDAC is highly expressed in the brain of the transgenic mice withAD-like disease. FIG. 15 shows cross-sections of brain from wild type(WT) and 5XFAD transgenic (TG) mice (FIG. 15A) and immuno-staining ofbrain sections from WT and TG mice with anti-Aβ or anti-VDAC1 antibodies(FIG. 15B).

The compound of Formula 1 (VBIT-4) was dissolved in drinking water asfollows: about 24 mg of VBIT-4 were transferred to Eppendorf tube anddissolved in 120 μl of 100% DMSO by Vortex mixer. Clear solution wasobtained. About 10 mL solution of 1 M of HCl was prepared from 6 M HClsolution, provided by Pierce, Rockford, Ill., USA. About 370 μL of the1M HCl solution was used to acidify 120 mL of drinking water. The VBIT-4DMSO solution (120 μL) was slowly added (by dropping) into the acidicwater and mixed by magnetic stirring. The final pH was between 4.8 and5.0. If the solution became milky, further 10 to 30 μL of HCl solutionwere added to obtain clear solution. The amount was sufficient for 24mice at dose of 20 mg/kg and drinking volume of 5 mL per mouse per day.

Animals at age of two months were assigned to three groups: transgenictreated (TG-T, 8 males and 3 females), transgenic vehicle (TG-V, 8 malesand 3 females), and wild type (WT, 10 males and 8 females). Of these, 2males in TG-T group died during the study.

Two-month old 5XFAD mice were provided with 0.9% of DMSO solution orVBIT-4 solution (20 mg/kg in 0.9% DMSO) in drinking water, replaced withfresh solution three times a week in the first month and thereaftertwice a week for additional 3 months.

When the mice reached the age of six months, a two-day radial-arm watermaze (RAWM) trial was performed as described previously (JenniferAlamed, et al, Nature Protocols 1. 2006. 1671-1679) to test the effectof VBIT-4 on learning and memory task. An RAWM containing six swim paths(arms) was used. The arms were extending out from an open central areawith an escape platform located at the end of one arm (the goal arm).The goal arm location remained constant for a given mouse. On day 1,mice were trained for 15 trials (spaced over 3 h), with trialsalternating between visible and hidden platforms. On day 2, mice weretrained for 15 trials with the hidden platform.

Entry into an incorrect arm was scored as an error, and the times spentby the animal to find the platform were recorded. The results aredemonstrated in FIGS. 14A and 14B. FIG. 14A shows the number of errors,while FIG. 14B shows the total time spend in the water maze, as functionof number of learning blocks. The number of errors data (FIG. 14A, datapresented as mean±standard error of the mean) from RAWM was assessedusing ANOVA test; a significant difference in animal's memory trainingbetween the three groups: WT-mice (n=18), 5XFAD/APOE (n=13) and5XFAD/APOE treated with VBIT-4 (n=9) in different measurement times(trials) was obtained with F (9,159)=2.03 (p=0.03). To examine thesource of differences the post-hoc test of Bonferroni-type was used. Thenon-transgenic mice trained better in comparison to non-treated TG mice(p=0.007). The TG mice treated with VBIT-4 performance was better thanuntreated TG and there was no difference between TG VBIT-4 treated groupand the WT group. A trend for the improved performance (training) of theVBIT-4-TG treated group compared to non-treated TG group was seen(p=0.06).

The data of total time spent (FIG. 14B, data presented as mean±standarderror of the mean) from RAWM was analyzed using repeated measures ANOVAtest; a significant difference was obtained in animal's memory trainingbetween the three mouse groups: WT (n=18), 5XFAD/APOE (n=13) and5XFAD/APOE treated with VBIT-4 (n=9) in different measurement times(trials), with F (2,35)=6.91, p=0.003. To examine the source ofdifferences the post-hoc test of Bonferroni-type was used. Thenon-transgenic mice trained learned better in comparison to non-treatedTG mice (p=0.003). The performance of TG mice treated with VBIT-4 wasbetter than untreated TG and there was no difference between TG VBIT-4treated group and the WT group.

To assess memory function, VBIT-4 was given to 5XFAD transgenic (TG)mice with AD-like disease (in drinking water) and the mice weresubjected to T-maze (FIG. 16a ) and Y-maze (FIG. 16b ) tests, after 6months of treatment. Wild type (WT) mice and TG mice treated with avehicle (0.9% DMSO solution) served as control.

T-Maze assay is based on the willingness of rodents to explore a newenvironment and allows for evaluating cognition, especially spatial andworking memory (Rosenmann, H., et al., 2008. Exp Neurol 212(1), p.71-84). Y-maze assay evaluates the willingness of the mice to explorenew environments and to assess memory function and is useful inevaluating the effects of drugs on cognition.

The TG mice treated with VBIT-4 showed improved learning and memorytasks, reaching levels of wild type mice, in several tests. Theperformance of the transgenic mice with AD-like disease treated withVBIT-4 was equivalent to the performance of wild type mice (FIG. 16).This suggests that VBIT-4 reaches its target at sufficientconcentrations to mediate its effects.

Example 7: Effect of Compound of Formula 1 on Schizophrenia

Startle response is comprised of a constellation of reflexes elicited bysudden relatively intense stimuli. It offers many advantages as abehavioral measure of central nervous system activity when elicited byacoustic (noise burst), electrical (cutaneous), tactile (air puff), orvisual (light flash) stimuli. The startle reflex has served as a toolfor studying fundamental properties of nervous function of complexbehavioral states and cognitive processes. The forebrain modulatesseveral forms of startle plasticity including the habituation andprepulse inhibition (PPI). Changes in startle magnitude through repeatedstimulus presentations-habituation and sensitization represent thesimple forms of learning. Quantification of startle habituation andsensitization in rodent has direct physiological relevance to human CNSfunction. In fact, the most well accepted animal physiology models forschizophrenia are startle habituation and PPI.

Startle magnitude is reduced when the pulse stimulus is preceded 30 to500 msec by a weak prepulse. This inhibition (“gating”) of a motorresponse elicited by a weak sensory event, termed PPI, provides anoperational measure of sensorimotor gating. Prepulse stimuli of 3, 6, or12 dB above the 70 dB background noise inhibit the startle responseelicited by 120-dB pulse stimuli. Prestimuli used in intramodal studiesof sensorimotor gating of acoustic startle are by the delivery of adiscrete acoustic prepulse several msec before the startle pulse, withintensity below startle threshold. Holding the interval between theprepulse and pulse stimuli constant at 100 msec, typically yieldssuitable levels of PPI, ranging from 20% to 80% inhibition.

The effect of the compound of Formula 1 (VBIT-4) and/or Formula 10(AKOS-022) and/or Formula 3 (VBIT-12) on induction of startlehabituation and PPI in animals is examined.

For habituation, six trials of a single acoustic stimulus are presentedto each mouse. To provide a consistent acoustic environment and to maskexternal noises, a continuous background noise level of 70 dB withineach startle chamber is maintained. The peak or average response fromeach mouse on each of six trials is collected, and then the sixresponses are averaged for each mouse. Five more trials at the end ofPPI are performed. The results are averaged and compared to the originalsix trials. The difference of startle responses between the initial sixand the last five trials are considered the amount of habituation.Analyses can include the independent variable (e.g., vehicle or drugtreatment) as a factor in analyses of variance (ANOVA) on the dependentmeasures (difference of the average of the first and last six trials).

The assay includes total of 36 trials. The three prepulse stimuli are ofduration of 20 msec. For each mouse the following metrics aredetermined: 1) Average response magnitude on pulse-only, trials 1 to 6and 32 to 36; 2) Average response magnitude in each of the four trialtypes between trials 7 and 31 inclusively (i.e., ten pulse-only trialsand five each of the three prepulse variations). The first block ofpulse-only trials are analyzed as measures of startle reactivity. Thefirst and last blocks of pulse-only trials are analyzed together in arepeated measure ANOVA to assess habituation of acoustic startle acrossthe test session. The four values (3, 6, or 12 dB above background)derived from trials 7 to 31 are used to assess PPI which is calculatedfor each mouse as: Percentage score: PPI=100%×{[pulse-onlyunits−(prepulse+pulse units)]/(pulse-only units)}.

Male 129 SVE adult mice are first tested at baseline. Four groups ofmice (typically 10 mice in each group) are treated with the compounds ofFormulae 1 (VBIT-4), Formula 10 (AKOS-022), Formula 3 (VBIT-12) or withvehicle (0.9% DMSO). The mice receive the compounds in drinking water asdescribed in Example 6 above at 20 mg/kg throughout the experiment.

Amphetamine is a drug that may induce psychosis, and amphetamine-inducedpsychosis is used as a model for primary psychotic disorders.Specifically, amphetamine induces disruption of habituation. Amphetamine(10 mg/kg) is administered 30 minutes before the experiments. The effectof VBIT-4 and/or AKOS-022 and/or VBIT-12 on habituation disruption byamphetamine is measured.

Example 8: Effects of the Compounds of Formula 1 (VBIT-4) or Formula 10(AKOS-022) on Depression

Male BALB/c mice aged 8 weeks are divided to two groups: a control groupreceiving vehicle solution (0.9% DMSO) and a treatment group receiving asolution of the compound of Formula 1 (VBIT-4) and/or the compound ofFormula 10 (AKOSS-022) and/or the compound of Formula 3 (VBIT-12) in thedrinking water as described in Example 6 hereinabove. The compound isprovided at 20 mg/kg dissolved in 0.9% DMSO.

Novelty Suppressed Feeding Test

After the last treatment is administered, the mice do not receive anyfood for 24 hours (water was available ad libitum). At the end of thisperiod, each animal is introduced into a 50×50×30 (height) cm plasticarena located in a specialized behavioral room in which all behavioraltests were tracked by cameras. The cameras may be linked to a computerinstalled with behavioral tracking software, for example EthoVision (XT10). A pellet of food is placed on an elevated surface in the center ofthe arena. Time elapsing from the introduction of the animal into thearena until it commences eating (latency to feed), total distance move,velocity and time spent in the arena periphery (lateral 10 cm on eachside) and center are recorded. The animal is then removed from the arenaimmediately after it begins to eat or after not doing so for 5 min.After the test, the animal is immediately transferred to its home cageand left to consume a previously weighed amount of food for 10 minutes.On completion of this period the food is weighed again to calculate thehome cage food consumption. The rating of the animals' behavior isconducted by two experimenters who are blind to the treatment receivedby each mouse. The mean of the two ratings is calculated and used forthe statistical analysis.

Sweet Preference Model of Anhedonia and Forced Swim Test Model forDespair

Sweet Preference

Mice are housed individually in cages having two 200 ml bottlescontaining tap water for 5 days. For the next 6 days, one of the bottlesis replaced with a bottle containing a 5% sucrose solution and the otheris left to contain tap water. The two bottles are presented for 12 h inthe dark phase, following 12 h deprivation in the light phase, switchingthe sides on day 3.

Body weight is monitored weekly and fluid intake monitored daily.

Sucrose preference is calculated as the percent of the total fluidintake.

Forced Swim Test (FST)

Forced Swim Test (FST) is an animal model used to assess antidepressantcompounds in preclinical studies. In this paradigm, mice are forced toswim in an inescapable cylinder filled with water. Under theseconditions, mice will initially try to escape and eventually developimmobility behavior; this behavior is interpreted as a passivestress-coping strategy or depression-like behavior.

The assay is performed in swim tanks (for example, glass cylinders (46cm height×20 cm diameter) containing water (20-cm deep, maintained at24-25° C.).

One week after the sweet preference test, the mice are tested in theFST. The test compound is administered to each mice 15-30 min beforeswim session, e.g. by i.v. route. Each mouse is filmed and scoredblindly after first establishing reliability between two blind observers(r=0.94) in the last 4 min as described in Lin et al. (Lin T, et al.,2012. Int J Dev Neurosci. 3((2), 113-20). The dependent measures were:(1) Immobility: No movement except for minimal paw and tail movementsnecessary to keep afloat, (2) Climbing: rhythmic bilateral movements offorelimbs and hindlimbs with body stretched vertically along theperiphery of the container, and (3) Swimming which was calculated as thedifference between 4 min and the time spent in immobility plus climbing.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

What is claimed is:
 1. A method for treating a CNS-associated disorderselected from the group consisting of psychotic disorder, mood disorderand neurodegenerative disease, the method comprising the step ofadministering to a subject in need thereof at least one compound of thegeneral formula (I):

wherein: A is carbon (C) or nitrogen (N); R³ is absent, a hydrogen, anunsubstituted or substituted amide, or a heteroalkyl comprising 3-12atoms (apart from hydrogen atoms), wherein at least one atom is anitrogen, sulfur or oxygen atom, wherein when A is nitrogen (N), R³ isabsent; L¹ is absent or is an amino linking group —NR⁴—, wherein R⁴ ishydrogen, a C₁₋₅-alkyl, a C₁₋₅-alkylene or a substituted alkyl —CH₂R,wherein R is a functional group selected from the group consisting ofhydrogen, halo, haloalkyl, cyano, nitro, hydroxyl, alkyl, alkenyl, aryl,alkoxyl, aryloxyl, aralkoxyl, alkylcarbamido, arylcarbamido, amino,alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl and heteroaryl; R¹ is an aromatic moiety, which isoptionally substituted with one or more of Z; Z is independently at eachoccurrence a functional group selected from the group consisting of,hydrogen, halo, haloalkyl, haloalkoxy, perhaloalkoxy orC₁₋₂-perfluoroalkoxy, cyano, nitro, hydroxyl, alkyl, alkenyl, aryl,alkoxyl, aryloxyl, aralkoxyl, alkylcarbamido, arylcarbamido, amino,alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl and heteroaryl; L² is a linking group, such that when A isnitrogen (N), L² is a group consisting of 4-10 atoms, apart fromhydrogen atoms, optionally forming a ring, whereof at least one of theatoms is nitrogen, said nitrogen forming part of an amide group; andwhen A is carbon (C), then L² is selected from C₁₋₄ alkylene or a groupconsisting of 4-10 atoms, apart from hydrogen atoms, optionally forminga ring, whereof at least one of the atoms is nitrogen, said nitrogenforming part of an amide group; R² is a phenyl or a naphthyl, optionallysubstituted with halogen; or an enantiomer, diastereomer, mixture orsalt thereof.
 2. The method of claim 1, wherein A is nitrogen (N), andsaid linking group L² is selected from the group consisting of aC₄₋₆-alkylamidylene and a pyrrolidinylene, said linking group optionallysubstituted with one or two of alkyl, hydroxy, oxo or thioxo group. 3.The method of claim 2, wherein L² is selected from the group consistingof butanamidylene, N-methylbutanamidylene, N,N-dimethylbutanamidylene,4-hydroxybutanamidylene, 4-oxobutanamidylene,4-hydroxy-N-methylbutanamidylene, 4-oxo-N-methylbutanamidylene,2-pyrrolidonyl, pyrrolidine-2,5-dionylene, 5-thioxo-2-pyrrolidinonyleneand 5-methoxy-2-pyrrolidinonylene.
 4. The method of claim 3, whereinwhen L² is butanamidylene, N-methylbutanamidylene,N,N-dimethylbutanamidylene, 4-hydroxybutanamidylene,4-oxobutanamidylene, 4-hydroxy-N-methylbutanamidylene or4-oxo-N-methylbutanamidylene, the carbon (C) in third position of thebutanamide moiety is bonded to the nitrogen (N) of the piperazine ringand the nitrogen (N) of the butanamide moiety is bonded to R²; orwherein when L² is 2-pyrrolidone, pyrrolidine-2,5-dione,5-thioxo-2-pyrrolidone or 5-methoxy-2-pyrrolidone, a carbon (C) of thepyrrolidine moiety is bonded to the nitrogen (N) of the piperazine ringand the nitrogen (N) of the pyrrolidine moiety is bonded to R².
 5. Themethod of claim 1, wherein A is carbon (C), R³ is a heteroalkyl group,and L² is methylene.
 6. The method of claim 1, wherein the compound isof general Formula (Ia):

wherein: A, R³, Z and L¹ as defined in claim 1, L^(2′) is a linkinggroup selected from the group consisting of an C₄-alkylamidylene,C₅-alkylamidylene and C₆-alkylamidylene, optionally substituted with oneor two of alkyl, hydroxy, oxo or thioxo group; and Y is halogen; or anenantiomer, diastereomer, mixture or salt thereof.
 7. The method ofclaim 6, wherein carbon (C) at position 3 of alkylamidylene L^(2′) isbonded to the nitrogen (N) of the piperazine ring or of the piperidinering, and the nitrogen (N) of said alkylamidylene L^(2′) is bonded tothe phenyl group.
 8. The method of claim 1, wherein the compound is ofthe general formula (Ib):

wherein: A, R³, and Z are as defined in claim 1, L¹ is absent; L^(2″) isa pyrrolidinylene linking group, optionally substituted with one or twoof alkyl, hydroxy, oxo or thioxo group; Y is halogen; or an enantiomer,diastereomer, mixture or salt thereof.
 9. The method of claim 8, whereinL^(2″) is selected from 2-pyrrolidonylene, pyrrolidine-2,5-dionylene,5-thioxo-2-pyrrolidinonylene and 5-methoxy-2-pyrrolidinonylene.
 10. Themethod of claim 8, wherein a carbon (C) atom of the pyrrolidinyl moietyL2″ is bonded to the nitrogen (N) of the piperazine ring or thepiperidine ring and the nitrogen (N) of the pyrrolidinyl moiety isbonded to the phenyl group.
 11. The method of claim 1, wherein thecompound is of the general formula (Ic):

wherein: A, R³, and Z are as defined in claim 1, L¹ is —NH—; Y¹ and Y²are each independently absent or a halogen; or an enantiomer,diastereomer, mixture or salt thereof.
 12. The method of claim 11,wherein R³ is —C(O)NHCH₂C(O)OH group.
 13. The method of claim 1, whereinZ is C₁₋₂-alkoxy or C₁₋₂-perfluoroalkoxy.
 14. The method of claim 2,wherein the compound is of the general formula (Id):

wherein Z is C₁₋₂-perfluoroalkoxy, and Y is halogen.
 15. The method ofclaim 14, wherein the compound is selected from the group consisting ofa compound having the Formula 1:

and a compound having the Formula 2:

or an enantiomer, diastereomer, mixture or salt thereof.
 16. The methodof claim 12, wherein the compound having the Formula 3:

or an enantiomer, diastereomer, mixture or salt thereof.
 17. The methodof claim 1, wherein the compound is of Formula (IIa):

wherein: A is carbon (C); R³ is hydrogen or heteroalkyl chain comprising3-12 atoms, apart from hydrogen atoms, wherein at least one is aheteroatom, selected from nitrogen, sulfur and oxygen; L¹ is an aminolinking group —NR⁴—, wherein R⁴ is hydrogen, a C₁₋₅-alkyl, aC₁₋₅-alkylene or a substituted alkyl —CH₂R, wherein R is a functionalgroup selected from hydrogen, halo, haloalkyl, cyano, nitro, hydroxyl,alkyl, alkenyl, aryl, alkoxyl, aryloxyl, aralkoxyl, alkylcarbamido,arylcarbamido, amino, alkylamino, arylamino, dialkylamino, diarylamino,arylalkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,alkylcarbonyloxy, arylcarbonyloxy, carboxyl, alkoxycarbonyl,aryloxycarbonyl, sulfo, alkylsulfonylamido, alkylsulfonyl, arylsulfonyl,alkylsulfinyl, arylsulfinyl or heteroaryl; when R³ is heteroalkyl groupcomprising 3-12 atoms, apart from hydrogen atoms, then L¹ forms a ringwith R³; R¹ is an aromatic moiety, which is optionally substituted withone or more of C₁₋₂-alkoxy, and/or C₁₋₂-perfluoroalkoxy; L² is a linkinggroup consisting of 4-10 atoms, apart from hydrogen atoms, optionallyforming a ring, whereof at least one of the atoms is nitrogen, saidnitrogen forming part of an amide group or L² is C₁₋₅ alkyl or C₁₋₅alkylene; said linking group L² bonds piperidine or piperazine moiety atnitrogen (N) atom; and R² is an aryl, optionally substituted withhalogen, optionally when R² is a phenyl it is substituted with halogen,further optionally when R² is naphthyl, L² is an alkylenyl group; or anenantiomer, diastereomer, mixture or salt thereof.
 18. The methodaccording claim 17, wherein the compound of Formula (IIa) has theFormula 10:


19. The method according claim 18, wherein the compound of Formula (IIa)has the Formula 11:


20. The method of claim 1, wherein the compound is administered within apharmaceutical composition further comprising pharmaceuticallyacceptable excipients, diluents or carriers.
 21. The method of claim 1,wherein: the psychotic disorder is schizophrenia; the mood disorder isselected from the group consisting of bipolar disorder, major depressivedisorder, persistent depressive disorder and anxiety disorder; or theneurodegenerative disease is selected from the group consisting ofAlzheimer's disease, Parkinson disease and Amyotrophic Lateral Sclerosis(ALS).